Modified rapid expansion methods (“modified-REM”) for in vitro propagation of T lymphocytes

ABSTRACT

The present invention provides a modified rapid expansion method (termed “low-PBMC-REM” or “modified-REM”), for quickly generating large numbers of T lymphocytes, including cytolytic and helper T lymphocytes, without using the large excesses of peripheral blood mononuclear cells (PBMC) or EBV-transformed lymphoblastoid cells (LCL) characteristic of high-PBMC-REM. Clonal expansions of greater than 500-fold can be achieved within a single stimulation cycle of about 8-14 days.

This application is a national phase filing under 35 USC § 371 ofInternational Patent Application No. PCT/US97/03293, filed Mar. 3, 1997,which claims priority to U.S. Provisional Application No. 60/037,333,which was converted from U.S. patent application Ser. No. 08/610,710,filed Mar. 4, 1996.

FIELD OF THE INVENTION

This invention relates to improved methods for culturing T lymphocytes,including human antigen-specific cytolytic and helper T lymphocytes. Themethods of the present invention result in the very rapid and efficientexpansion of T cells which are useful, for example, in cellularimmunotherapy.

BACKGROUND

T lymphocytes are formed in the bone marrow, migrate to and mature inthe thymus and then enter the peripheral blood and lymphaticcirculation. T lymphocytes can be phenotypically subdivided into severaldistinct types of cells including: helper T cells, suppressor T cells,and cytotoxic T cells. T lymphocytes, unlike B lymphocytes, do notproduce antibody molecules, but express a heterodimeric cell surfacereceptor that can recognize peptide fragments of antigenic proteins thatare attached to proteins of the major histocompatibility complex (MHC)expressed on the surfaces of target cells; see, e.g., Abbas, A. K.,Lichtman, A. H., and Pober, J. S., Cellular and Molecular Immunology,1991, esp. pages 15-16.

T lymphocytes that can be expanded according to the present inventionare of particular interest in the context of cellular “immunotherapy”.As used herein, cellular immunotherapy refers to any of a variety oftechniques involving the introduction of cells of the immune system,especially T lymphocytes, into a patient to achieve a therapeuticbenefit. Such techniques can include, by way of illustration,“immuno-restorative” techniques (involving, e.g., the administration ofT cells to a patient having a compromised immune system);“immuno-enhancing” techniques (involving, e.g., the administration of Tcells to a patient in order to enhance the ability of that patient'simmune system to avoid or combat a cancer or a pathogen such as a virusor bacterial pathogen); and “immuno-modulating” techniques (involving,e.g., the administration of T cells to a patient in order to modulatethe activity of other cells of the patient's immune system, such as in apatient affected by an autoimmune condition).

Cytotoxic T lymphocytes (CTLs) are typically of the CD3+, CD4−, CD8+phenotype and lyse cells that display fragments of foreign antigensassociated with class I MHC molecules on their cell surfaces. CTLs thatare CD3+, CD4+, CD8− have also been identified. Target cells for CTLrecognition include normal cells expressing antigens after infection byviruses or other pathogens; and tumor cells that have undergonetransformation and are expressing mutated proteins or areover-expressing normal proteins.

Most “helper” T cells are CD3+, CD4+, CD8−. Helper T cells recognizefragments of antigens presented in association with class II MHCmolecules, and primarily function to produce cytokines that amplifyantigen-specific T and B cell responses and activate accessory immunecells such as monocytes or macrophages. See, e.g., Abbas, A. K., et al.,supra. Helper T cells can also participate in and/or augment cytolyticactivites.

In addition to conventional helper T cells and cytolytic or “killer” Tcells, it will also be useful to be able to rapidly expand other T cellpopulations. For example, T cells expressing the gamma/delta T cellreceptor represent a relatively small portion of the human T cellpopulation, but are suspected to play a role in reactivity to viral andbacterial pathogens as well as to tumor cells (see, e.g., W. Haas et al.1993. Annu. Rev. Immunol. 11:637). Another T cell population ofpotential clinical importance is the population of CD1-restricted Tcells. CD1 is an MHC-like molecule that shows limited polymorphism and,unlike classical MHC molecules which “present” antigenic peptides, CDmolecules bind lipoglycans and appear to be important in the recognitionof microbial antigens (see, e.g., P. A. Sieling et al. 1995. Science269:227; and E. M. Beckman et al. 1994. Nature 372:691).

T lymphocytes are thus key components of the host immune response toviruses, bacterial pathogens and to tumors. The significance of properlyfunctioning T cells is made quite clear by individuals with congenital,acquired or iatrogenic T cell immunodeficiency conditions (e.g., SCID,BMT, AIDS, etc.) which can result in the development of a wide varietyof life-threatening infections or malignancies. Persons with diseasesthat are related to a deficiency of immunologically-competent Tlymphocytes, or persons with conditions that can be improved byadministering additional T lymphocytes, can thus be benefited bycellular immunotherapies, as referred to above. T cells for use in suchtherapies can be derived from the immunodeficient host, or from anothersource (preferably a compatible donor). The latter source is of courseespecially important in situations in which an immunodeficient host hasan insufficient number of T cells, or has T cells that areinsufficiently effective. In either case, it is difficult to obtainsufficient numbers of T cells for effective administration; and thustarget T cells must first be grown to large numbers in vitro beforeadministration to a host.

After undergoing such cellular immunotherapy, hosts that previouslyexhibited, e.g., inadequate or absent responses to antigens expressed bypathogens or tumors, can express sufficient immune responses to becomeresistant or immune to the pathogen or tumor.

Adoptive transfer of antigen-specific T cells to establish immunity hasbeen demonstrated to be an effective therapy for viral infections andtumors in animal models (reviewed in Greenberg, P. D., Advances inImmunology (1992)). For adoptive immunotherapy to be effective,antigen-specific T cells usually need to be isolated and expanded innumbers by in vitro culture, and following adoptive transfer suchcultured T cells must persist and function in vivo. For treatment ofsome human diseases, the use in immunotherapy of cloned antigen-specificT cells which represent the progeny of single cells, offers significantadvantages because the specificity and function of these cells can berigorously defined and precise dose:response effects readily evaluated.Riddell et al. were the first to adoptively transfer humanantigen-specific T cell clones to restore deficient immunity in humans.Riddell, S. R. et al., “Restoration of Viral Immunity in ImmunodeficientHumans by the Adoptive Transfer of T Cell Clones”, Science 257:238-240(1992). In that study, Riddell et al. used adoptive immunotherapy torestore deficient immunity to cytomegalovirus in allogeneic bone marrowtransplant recipients. Cytomegalovirus-specific CD8+ cytotoxic T cellclones were isolated from three CMV seropositive bone marrow donors,propagated in vitro for 5 to 12 weeks to achieve numerical expansion ofeffector T cells, and then administered intravenously to the respectivebone marrow transplant (BMT) recipients. The BMT recipients weredeficient in CMV-specific immunity due to ablation of host T cellresponses by the pre-transplant chemoradiotherapy and the delay inrecovery of donor immunity commonly observed after allogeneic bonemarrow transplant (Reusser et al. Blood, 78:1373-1380, 1991). Riddell etal. found that no toxicity was encountered and that the transferred Tcell clones provided these immunodeficient hosts with rapid andpersistent reconstitution of CD8+ cytomegalovirus-specific CTLresponses.

Riddell et al. (J. Immunology, 146:2795-2804, 1991) used the followingprocedure for isolating and culturing the CD8+ CMV-specific T cellclones: peripheral blood mononuclear cells (PBMCs) derived from the bonemarrow donor were first cultured with autologouscytomegalovirus-infected fibroblasts to activate CMV-specific CTLprecursors. Cultured T cells were then restimulated with CMV-infectedfibroblasts and the cultures supplemented with γ-irradiated PBMCs. 2-5U/ml of interleukin-2 (IL-2) in suitable culture media was added on days2 and 4 after restimulation to promote expansion of CD8+ CTL (Riddell etal., J. Immunol., 146:2795-2804, 1991). To isolate T cell clones, thepolyclonal CD8+ CMV-specific T cells were plated at limiting dilution(0.3-0.6 cells/well) in 96-well round bottom wells with eitherCMV-infected fibroblasts as antigen-presenting cells (Riddell, J.Immunol., 146:2795-2804, 1991); or anti-CD3 monoclonal antibody to mimicthe stimulus provided by antigen-presenting cells. (Riddell, J. Imm.Methods, 128:189-201, 1990). Then, γ-irradiated peripheral bloodmononuclear cells (PBMC) and EBV-transformed lymphoblastoid cells (LCL)were added to the microwells as feeder cells. Wells positive for clonalT cell growth were evident in 10-14 days. The clonally derived cellswere then propagated to large numbers initially in 48- or 24-well platesand subsequently in 12-well plates or 75-cm² tissue culture flasks. Tcell growth was promoted by restimulation every 7-10 days withautologous CMV-infected fibroblasts and γ-irradiated feeder cellsconsisting of PBMC and LCL, and the addition of 25-50 U/ml of IL-2 at 2and 4 days after restimulation.

A major problem that exists in the studies described above, and ingeneral in the prior art of culturing T cells, is the inability to growlarge quantities of human antigen-specific T cell clones in a timelyfashion. It is not known if the slow growth of T cells in culturerepresents an inherent property of the cell cycle time for humanlymphocytes or the culture conditions used. For example, with theculture method used in the CMV adoptive immunotherapy study describedabove, three months were required to grow T cells to achieve the highestcell dose under study which was 1×10⁹ T cells/cm². This greatly limitsthe application of adoptive immunotherapy for human viral diseases andcancer since the disease process may progress during the long intervalrequired to isolate and grow the specific T cells to be used in therapy.Based on extrapolation from animal model studies (reviewed in Greenberg,P. D., Advances in Immunology, 1992), it is predicted that in humansdoses of antigen-specific T cells in the range of 10⁹-10¹⁰ cells may berequired to augment immune responses for therapeutic benefit.

However, rapidly expanding antigen-specific human T cells in culture toachieve such high cell numbers has proven to be a significant obstacle.Thus, with the exception of the study by Riddell et al., supra, (inwhich several months were taken to grow a sufficient number of cells)studies of adoptive immunotherapy using antigen-specific T cell cloneshave not been performed. The problem of producing large numbers of cellsfor adoptive immunotherapy was identified in U.S. Pat. No. 5,057,423. Inthis patent, a method for isolating pure large granular lymphocytes anda method for the expansion and conversion of these large granularlymphocytes into lymphokine activated killer (LAK) cells is described.The methods are described as providing high levels of expansion, i.e. upto 100-fold in 3-4 days of culture. Although LAK cells will lyse sometypes of tumor cells, they do not share with MHC-restricted T cells theproperties of recognizing defined antigens and they do not provideimmunologic memory. Moreover, the methods used to expand LAK cells,which predominantly rely on high concentrations of IL-2 do notefficiently expand antigen-specific human T cells (Riddell et al.,unpublished); and those methods can render T cells subject to programmedcell death (i.e. apoptosis) upon withdrawal of IL-2 or subsequentstimulation via the T cell receptor (see the discussion of the papers byLenardo et al, and Boehme et al., infra). Earlier methods that relied onthe use of lectins, such as concanavalin A or phytohemagglutinin (see,e.g., Van de Griend et al., Transplantation 38: 401-406 (1984), and Vande Griend et al., J. Immunol. Methods 66: 285-298 (1984)), are even lesssatisfactory because the use of such non-specific stimultory lectinstends to induce a number of phenotypic changes in the stimulated cellsthat make them quite different from T cells stimulated via the CD3receptor.

The inability to culture antigen-specific T cell clones to large numbershas in part been responsible for limiting adoptive immunotherapy studiesfor human diseases such as cancer (Rosenberg, New Engl. J. Med.,316:1310-1321, 1986; Rosenberg, New Engl. J. Med., 319:1676-1680, 1988)and HIV infection (Ho M. et al., Blood 81:2093-2101, 1993) to theevaluation of activated polyclonal lymphocyte populations with poorlydefined antigen specificities. In such studies, polyclonal populationsof lymphocytes are either isolated from the blood or the tumor filtrateand cultured in high concentrations of the T cell growth factor IL-2. Ingeneral, these cells have exhibited little if any MHC-restrictedspecificity for the pathogen or tumor and in the minority of patientsthat have experienced therapeutic benefit, it has been difficult todiscern the effector mechanism involved. Typically, adoptiveimmunotherapy studies with non-specific effector lymphocytes haveadministered approximately 2×10¹⁰ to 2×10¹¹ cells to the patient. (See,e.g., U.S. Pat. No. 5,057,423, at column 1, lines 40-43).

The development of efficient cell culture methods to rapidly grow Tlymphocytes will be useful in both diagnostic and therapeuticapplications. In diagnostic applications, the ability to rapidly expandT cells from a patient can be used, for example, to quickly generatesufficient numbers of cells for use in tests to monitor the specificity,activity, or other attributes of a patient's T lymphocytes. Moreover,the capability of rapidly achieving cell doses of 10⁹-10¹⁰ cells willgreatly facilitate the applicability of specific adoptive immunotherapyfor the treatment of human diseases.

There are several established methods already described for culturingcells for possible therapeutic use including methods to isolate andexpand T cell clones. Typical cell culture methods foranchorage-dependent cells, (i.e., those cells that require attachment toa substrate for cell proliferation) are limited by the amount of surfacearea available in culture vessels used (i.e., multi-well plates, petridishes, and culture flasks). For anchorage-dependent cells, the only wayto increase the number of cells grown is generally to use larger vesselswith increased surface area and/or use more vessels. However,hematopoietic cells such as T lymphocytes are anchorage-independent.They can survive and proliferate in response to the appropriate growthfactors in a suspension culture without attachment to a substrate. Evenwith the ability to grow antigen-specific lymphocytes in a suspensionculture, the methods reported to date have not consistently producedrapid numerical expansion of T cell clones. For example, in a study of Tcells conducted by Gillis and Watson, it was found that T cells culturedat low densities, i.e., 5×10³ to 1×10⁴ cell/ml in over a seven dayperiod and eventually reached a saturation density of 3-5×10⁵ cells/ml.Gillis, S. and Watson, J. “Interleukin-2 Dependent Culture of CytolyticT Cell Lines”, Immunological Rev., 54:81-109 (1981). Furthermore, Gillisand Watson also found that once cells reached this saturationconcentration, the cells would invariably die. Gillis et al., id.

Another study reported three different methods for establishing murine Tlymphocytes in long-term culture. Paul et al., reported that the methodmost widely used is to grow T lymphocytes from immunized donors forseveral weeks or more in the presence of antigen and antigen-presentingcells (APCs) to provide the requisite T cell receptor signal andco-stimulatory signals, and with the addition of exogenous growthfactors before attempting to clone them, Paul, W. E., et al., “Long-termgrowth and cloning of non-transformed lymphocytes”, Nature, 294:697-699,(1981). T cells specific for protein antigens are then cloned bylimiting dilution with antigen and irradiated spleen cells as a sourceof APCs. A second method involved growing T cells as colonies in softagar as soon as possible after taking the cells from an immunized donor.The T cells were stimulated in an initial suspension culture withantigen and a source of APCs, usually irradiated spleen cells. In thissecond approach, it was found that, after 3 days, the cells weredistributed in the upper layer of a two-layer soft agar culture system.The colonies were picked from day 4 to 8 and then expanded in long-termcultures. The third approach involved selecting cells for theirfunctional properties rather than their antigenic specificity and thengrowing them with a series of different irradiated feeder cells andgrowth factor containing supernatants. Paul, W. E. et al., “Long-termgrowth and cloning of non-transformed lymphocytes”, Nature, 294:697-699,(1981). It is apparent that with each of these methods, it is notpossible to expand individual T cell clones from a single cell to10⁹-10¹⁰ cells in a timely manner. Thus, despite the ability to cloneantigen-specific T cells, and convincing evidence of the therapeuticefficacy of T cell clones in accepted animal models, the technicaldifficulty in culturing human T cells to large numbers has impeded theclinical evaluation and application of cellular immunotherapeuticprocedures.

Yet another concern with cultured T cells is that they must remaincapable of functioning in vivo in order to be useful inimmunotherapeutic procedures. In particular, it has been observed thatantigen-specific T cells which were grown long term in culture in highconcentrations of IL-2 may develop cell cycle abnormalities and lose theability to return to a quiescent phase when IL-2 is withdrawn. Incontrast, the normal cell cycle consists of four successive phases:mitosis (or “M” phase) and three phases which make up the “interphase”stage. During the M phase, the cell undergoes nuclear division andcytokinesis. The interphase stage consists of the G1 phase in which thebiosynthetic activities resume at a high rate after mitosis; the S phasein which DNA synthesis occurs and the G2 phase which continues untilmitosis commences. While in the G1 phase, some cells appear to ceaseprogressing through the division cycle; and are said to be in a“resting” or quiescent state (denoted as the “G0” state). Certainenvironmental factors (such as a lack of growth factors in serum orconfluence of cell cultures) may cause cells to enter the quiescentstate. Once the factor is restored, the cell should resume its normalprogress through the cycle. However, cells grown in culture may beunable to enter the quiescent phase when the growth factor is removed,resulting in the death of these cells. This growth factor dependence isparticularly relevant to cultured T cells. T lymphocytes that areexposed over a long term to high concentrations of IL-2 to promote cellgrowth often will die by a process called apoptosis if IL-2 is removedor if they are subsequently stimulated through the T cell receptor,i.e., if they encounter specific antigens. (see, e.g., Lenardo M. J.,Nature, 353:858-861, 1991; Boehme S. A. and Lenardo M. J., Eur. J.Immunol., 23:1552-1560, 1992). Therefore, the culture methods used topropagate LAK cells or TIL-cells, and prior methods to culture T cellswhich predominantly rely on high long-term concentrations of IL-2 topromote expansion in vitro, may render many of the cells susceptible toapoptosis, thus limiting or eliminating their usefulness for cellularimmunotherapy.

It may also be advantageous in cellular immunotherapy studies to usegene transfer methods to insert foreign DNA into the T cells to providea genetic marker, to facilitate evaluation of in vivo migration andsurvival of transferred cells, or to confer functions that may improvethe safety and efficacy of transferred T cells. An established methodfor stable gene transfer into mammalian cells is the use of amphotropicretroviral vectors (see, e.g., Miller A D, Current Topics inMicrobiology and Immunology, 158:1-24, 1992). The stable integration ofgenes into the target cell using retrovirus vectors requires that thecell be actively cycling, specifically that these cells transit M phaseof the cell cycle. Prior studies have introduced a marker gene into asmall proportion of polyclonal T cells driven to proliferate with highdoses of IL-2, and these cells were reinfused into humans as tumortherapy and provided a means of following the in vivo survival oftransferred cells. (Rosenberg et al. New Engl. J. Med., 323:570-578,1990). However, for human T cells (which cycle slowly when grown withstandard techniques) the efficiency of stable gene transfer is very low,in the range of 0.1-1% of T cells. (Springett C M et al. J. Virology,63:3865-69, 1989). Culture methods which more efficiently recruit thetarget T cells into the S and G2-M phases of the cell cycle may increasethe efficiency of gene modification using retrovirus-mediated genetransfer (Roe T. et al., EMBO J, 2:2099-2108, 1993), thus improving theprospects for using genetically-modified T cells in cellularimmunotherapy or using T cells to deliver defective genes in geneticdeficiency diseases.

The rapid expansion method described by S. Riddell et al. (in PCTPublication WO 96/06929, published Mar. 7, 1996), hereinafter referredto as “high-PBMC REM” or “hp-REM” was developed to provide functional,antigen-specific T cell clones for use in clinical adoptiveimmunotherapy protocols. The hp-REM protocol was designed to providemaximal T cell expansion in a limited amount of time without loss of Tcell function and specificity. Generally, the hp-REM protocol involvesthe steps of adding an initial T lymphocyte population to a culturemedium in vitro; adding to the culture medium a disproportionately largenumber of non-dividing peripheral blood mononuclear cells (“PBMC”) asfeeder cells such that the resulting population of cells contains atleast about 40 PBMC feeder cells (preferably at least about 200, morepreferably at least about 400) for each T lymphocyte in the initialpopulation to be expanded; and incubating the culture. In preferredembodiments of the hp-REM protocol, the T cells to be expanded are alsoexposed to a disproportionately large number of EBV-transformedlymphoblastoid cells (“LCL”), to an anti-CD3 monoclonal antibody (e.g.,OKT3) (to activate the T cells via the T cell antigen receptor), and tothe T cell growth factor interleukin-2 (IL-2).

In the hp-REM protocol, T cells are generally expanded using a vastexcess of feeder cells consisting of peripheral blood mononuclear cells(PBMC) and possibly also EBV-transformed lymphoblastoid cells (EBV-LCL).T cells to be expanded typically represent less than about 0.2% of thecells in the hp-REM culture method. As described, the T cells can beactivated through the T cell antigen receptor using an anti-CD3monoclonal antibody (e.g. OKT3) and T cell proliferation can be inducedusing IL-2. Such hp-REM culture conditions were reported to result in alevel of T cell expansion 100 to 200-fold greater than that reported byothers.

However, for most uses, it would be preferable to avoid the use of largeexcesses of feeder cells (i.e. PBMC and EBV-LCL) in the preparation of Tcells destined for clinical use. For example, PBMCs are derived fromhuman blood and could represent a potential source of adventitiousagents (e.g. human imunodeficiency virus, type 1 and 2; human T cellleukemia virus I, type 1 and 2; and hepatitis virus, such as hepatitisB, C and G), and EBV-LCL could represent a potential source ofEpstein-Barr virus. In addition, the large-scale application of thehp-REM protocol would require a large supply of human peripheral bloodto provide adequate numbers of feeder cells.

It would therefore be particularly advantageous to reduce the numbers ofsuch feeder cells required or to replace them entirely. With theseconcerns in mind, the methods of the present invention (hereinafterreferred to as “low-PBMC-REM” or “modified-REM”) are designed to achieverapid in vitro expansion of T cells without using the vast excess ofPBMC and/or EBV-LCL feeder cells that are the key characteristic of thehp-REM protocol.

BRIEF SUMMARY OF THE INVENTION

This invention provides a method for rapidly producing large numbers ofT cells, including human antigen-specific cytolytic and helper T cells,isolated from an initial population of T cells, without using the vastexcess of PBMC and/or EBV-LCL feeder cells that are the keycharacteristic of the hp-REM protocol. While the methods of the presentinvention are applicable to the rapid expansion of T lymphocytes,generally, the rapid expansion method will be especially advantageous insituations in which an individual T cell clone must be expanded toprovide a large population of T lymphocytes. Thus, the present inventionprovides an especially important tool in the context of human adoptiveimmunotherapy, as has been exemplified in studies (using hp-REM,described below) involving human bone marrow transplant recipients atthe Fred Hutchinson Cancer Research Center. The present invention alsoprovides a method to improve the efficiency of stable gene transfer intoT lymphocytes, as exemplified below.

Accordingly, one object of the invention is to rapidly expand Tlymphocytes to large numbers in vitro without using the vast excess ofPBMC and/or EBV-LCL feeder cells that are the key characteristic of thehp-REM protocol. Such rapidly expanded T cell populations can be used,inter alia, for infusion into individuals for the purpose of conferringa specific immune response, as exemplified herein. T cells that can beexpanded using the present invention include any of the various Tlymphocyte populations described herein (see, e.g., the discussion aboveregarding CTLs, helper T cells and other T lymphocytes, and thepotential uses of such cells in immunotherapeutic techniques).

Another object of the invention is to use the method to grow T cells ina manner which facilitates the stable introduction of foreign geneticmaterial which can be used to alter the function of T cells to be usedin cellular immunotherapies, as described above, or to otherwiseovercome for a defective or inadequate gene in the host.

A number of preferred embodiments of the present invention are describedin the following enumeration:

1. A method (referred to herein as “low-PBMC-REM” or “modified-REM”) forrapidly expanding an initial T lymphocyte population in culture mediumin vitro, comprising the steps of: adding an initial T lymphocytepopulation to a culture medium in vitro; adding to the culture medium anon-dividing mammalian cell line expressing at least oneT-cell-stimulatory component, wherein said cell line is not anEBV-transformed lymphoblastoid cell line (LCL); and incubating theculture. REM cultures will generally be incubated under conditions oftemperature and the like that are suitable for the growth of Tlymphocytes. For the growth of human T lymphocytes, for example, thetemperature will generally be at least about 25 degrees Celsius,preferably at least about 30 degrees, more preferably about 37 degrees.Descriptions of suitable media and other culture conditions arewell-known in the art, and are also exemplified herein.

2. A rapid expansion method according to the preceding item, whereinsaid T-cell-stimulatory component is selected from the group consistingof an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine.

3. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of an Fc-γ receptor, a cell adhesion-accessory molecule and acytokine, and wherein said initial T lymphocyte population is expandedat least 200-fold after an incubation period of less than about twoweeks.

4. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of an Fc-γ receptor, a cell adhesion-accessory molecule and acytokine, and wherein said initial T lymphocyte population is expandedat least 500-fold after an incubation period of less than about twoweeks.

5. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of an Fc-γ receptor, a cell adhesion-accessory molecule and acytokine, and wherein said initial T lymphocyte population is expandedat least 1000-fold after an incubation period of less than about twoweeks.

6. A rapid expansion method according to any of the preceding items,further comprising the step of adding anti-CD3 monoclonal antibody tothe culture medium wherein the concentration of anti-CD3 monoclonalantibody is at least about 1.0 ng/ml. Typically, a concentration ofabout 10 ng/ml is employed although much lower levels can be used, asillustrated below.

7. A rapid expansion method according to any of the preceding items,further comprising the step of adding IL-2 to the culture medium,wherein the concentration of IL-2 is at least about 10 units/ml.Typically, a concentration of about 25 units/ml is used. Preferably, theincubation is continued for at least about 9 days and wherein the stepof adding IL-2 to the culture medium is repeated after each 3-5 dayinterval. Typically, IL-2 is added on day 0, again on day 5 or 6, andagain on day 8 or 9.

8. A rapid expansion method according to any of the preceding items,wherein said mammalian cell line comprises at least one cell type thatis present at a frequency at least twice that found in human peripheralblood mononuclear cells (human PBMCs); preferably at least three times,at least ten times, or at least fifty times the frequency generallyfound in human PBMCs.

9. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of an Fc-γ receptor and a cell adhesion-accessory molecule.

10. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of a cell adhesion-accessory molecule and a cytokine.

11. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is selected from the groupconsisting of an Fc-γ receptor and a cytokine.

12. A rapid expansion method according to any of the preceding items,wherein said mammalian cell line expresses a cell adhesion-accessorymolecule.

13. A rapid expansion method according to any of the preceding items,wherein said cell adhesion-accessory molecule is selected from the groupconsisting of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58,CD72, fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.

14. A rapid expansion method according to any of the preceding items,wherein said mammalian cell line expresses a cytokine. Preferably thecytokine is an interleukin.

15. A rapid expansion method according to any of the preceding items,wherein said T-cell-stimulatory component is a molecule that binds toCD21.

16. A rapid expansion method according to any of the preceding items,wherein said cytokine is selected from the group consisting of IL-1,IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.

17. A rapid expansion method according to any of the preceding items,further comprising the step of adding a soluble T-cell-stimulatoryfactor to the culture medium.

18. A rapid expansion method according to any of the preceding items,wherein said soluble T-cell-stimulatory factor is selected from thegroup consisting of a cytokine, an antibody specific for a T cellsurface component, and an antibody specific for a component capable ofbinding to a T cell surface component.

19. A rapid expansion method according to any of the preceding items,wherein said soluble T-cell-stimulatory factor is a cytokine selectedfrom the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL- 12 andIL-15.

20. A rapid expansion method according to any of the preceding items,wherein said soluble T-cell-stimulatory factor is an antibody specificfor a T cell surface component, and wherein said T cell surfacecomponent is selected from the group consisting of CD4, CD8, CD11a, CD2,CD5, CD49d, CD27, CD28 and CD44.

21. A rapid expansion method according to any of the preceding items,wherein said soluble T-cell-stimulatory factor is an antibody specificfor a component capable of binding to a T cell surface component, andwherein said T cell surface component is selected from the groupconsisting of CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.

22. A rapid expansion method according to any of the preceding items,wherein said soluble T-cell-stimulatory factor is a molecule that bindsto CD21.

23. A rapid expansion method according to any of the preceding items,wherein said molecule that binds to CD21 is an anti-CD21 antibody.

24. A rapid expansion method according to any of the preceding items,further comprising the step of adding to the culture a multiplicity ofperipheral blood mononuclear cells (PBMCs). Preferably, PBMC areirradiated with gamma rays in the range of about 3000 to 3600 rads, morepreferably at about 3300 rads.

25. A rapid expansion method according to any of the preceding items,wherein the ratio of PBMCs to initial T cells to be expanded is lessthan about 40:1.

26. A rapid expansion method according to any of the preceding items,wherein the ratio of PBMCs to initial T cells to be expanded is lessthan about 10:1.

27. A rapid expansion method according to any of the preceding items,wherein the ratio of PBMCs to initial T cells to be expanded is lessthan about 3:1.

28. A rapid expansion method according to any of the preceding items,further comprising the step of adding to the culture a multiplicity ofEBV-transformed lymphoblastoid cells (LCLs). Preferably, PBMC areirradiated with gamma rays in the range of about 6000 to 10,000 rads,more preferably at about 8000 rads.

29. A rapid expansion method according to any of the preceding items,wherein the ratio of LCLs to initial T cells to be expanded is less thanabout 10:1.

30. A rapid expansion method according to any of the preceding items,wherein the initial T lymphocyte population comprises at least one humanCD8+ antigen-specific cytotoxic T lymphocyte (CTL). In preferredembodiments of the present invention, the CTL is specific for an antigenpresent on a human tumor or encoded by a pathogen such as a virus orbacterium.

31. A rapid expansion method according to any of the preceding items,wherein the initial T lymphocyte population comprises at least one humanCD4+ antigen-specific helper T lymphocyte.

32. A method of genetically transducing a human T cell, comprising thesteps of: adding an initial T lymphocyte population to a culture mediumin vitro; adding to the culture medium a non-EBV-transformed mammaliancell line expressing a T-cell-stimulatory component; and incubating theculture; and adding a vector to the culture medium. A vector refers to aunit of DNA or RNA in a form which is capable of being introduced into atarget cell. Transduction is used generally to refer to the introductionof such exogenous DNA or RNA into a target cell and includes theintroduction of heterologous DNA or RNA sequences into target cells by,e.g., viral infection and electroporation. A currently preferred methodof transducing T lymphocytes is to use retroviral vectors, asexemplified herein.

33. A genetic transduction method according to item 32, wherein thevector is a retroviral vector containing a selectable marker providingresistance to an inhibitory compound that inhibits T lymphocytes, andwherein the method further comprises the steps of: continuing incubationof the culture for at least one day after addition of the retroviralvector; and adding said inhibitory compound to the culture medium aftersaid continued incubation step. Preferably, the retroviral vectorcontains both a positive and a negative selectable marker. Preferredpositive selectable markers are derived from genes selected from thegroup consisting of hph, neo, and gpt, and preferred negative selectablemarkers are derived from genes selected from the group consisting ofcytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Especiallypreferred markers are bifunctional selectable fusion genes wherein thepositive selectable marker is derived from hph or neo, and the negativeselectable marker is derived from cytosine deaminase or a TK gene.

34. A genetic transduction method according to any of items 32-33,further comprising adding a multiplicity of human PBMCs.

35. A rapid expansion method according to any of items 32-34, whereinthe ratio of PBMCs to initial T cells is less than about 40:1.

36. A genetic transduction method according to any of items 32-35,further comprising adding non-dividing EBV-transformed lymphoblastoidcells (LCL).

37. A rapid expansion method according to any of items 32-36, whereinthe ratio of LCL to initial T cells is less than about 10:1.

38. A method of generating a REM cell line capable of promoting rapidexpansion of an initial T lymphocyte population in vitro, comprising thesteps of: depleting one or more cell types from a human PBMC populationto produce a cell-type-depleted PBMC population, using saidcell-type-depleted PBMC population in place of non-depleted PBMCs in anhp-REM protocol to determine the contribution of the depleted cell typeto the activity provided by the non-depleted PBMCs, identifying a T cellstimulatory activity provided by said depleted cell type, andtransforming a mammalian cell line with a gene allowing expression ofsaid T cell stimulatory activity.

39. A method of generating a REM cell line according to item 38, whereinsaid T-cell-stimulatory component is selected from the group consistingof an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine.

40. A REM cell line capable of stimulating rapid expansion of an initialT lymphocyte population in vitro, comprising a mammalian cell linegenerated according to a method according to the preceding item 38 oritem 39.

41. A REM cell line according to item 40, wherein said cell lineexpresses a cell adhesion-accessory molecule.

42. A REM cell line according to any of items 40-41, wherein said celladhesion-accessory molecule is selected from the group consisting ofClass II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72,fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.

43. A REM cell line according to any of items 40-42, wherein said cellline expresses an Fc-γ receptor.

44. A REM cell line according to any of items 40-43, wherein said cellline expresses at least one T cell stimulatory cytokine.

45. A REM cell line according to any of items 40-44, wherein said T cellstimulatory cytokine is selected from the group consisting of IL-1,IL-2, IL-6, IL-7, IL-12 and IL-15.

46. A REM cell line according to any of items 40-44, wherein said cellline expresses a molecule that binds CD21. As used herein, a moleculethat binds CD21 can be a natural or synthetic molecule known ordetermined to bind to the CD21 cell surface determinant. Molecules knownto bind to CD21 include anti-CD21 antibodies, as well as molecules suchas C3d, C3dg, iC3b and EBV gp350/220, and derivatives thereof.

47. A culture medium capable of rapidly expanding an initial Tlymphocyte population in vitro comprising a REM cell line according toany of items 40-46.

48. A culture medium according to item 47, further comprising anexogenous cytokine.

49. A culture medium according to any of items 47-48, further comprisinga multiplicity of exogenous cytokines, wherein said multiplicitycomprises at least one interleukin.

50. A culture medium according to any of items 47-49, wherein saidinterleukin is selected from the group consisting of IL-1, IL-2, IL-6,IL-7, IL-12 and IL-15.

51. A culture medium according to any of items 47-50, further comprisinga molecule that binds to CD21. As used herein, a molecule that bindsCD21 can be a natural or synthetic molecule known or determined to bindto the CD21 cell surface determinant. Molecules known to bind to CD21include anti-CD21 antibodies, as well as molecules such as C3d, C3dg,iC3b and EBV gp350/220, and derivatives thereof that bind to CD21.

52. A culture medium according to item 51, wherein said molecule thatbinds to CD21 is an anti-CD21 antibody.

53. A culture medium according to any of items 49-52, further comprisingan anti-CD3 monoclonal antibody.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND APPLICATIONS OF THEINVENTION

The invention described herein provides methods for rapidly expandingpopulations of T lymphocytes, including human cytotoxic T lymphocytesand helper T lymphocytes, which can be particularly useful in cellularimmunotherapy of human diseases, without using the vast excess of PBMCand/or EBV-LCL feeder cells that are the key characteristic of thehp-REM protocol.

The T cells will be referred to as “target T cells”. In general, targetT cells are added in small numbers to a culture vessel containingstandard growth medium that has been supplemented with components thatstimulate rapid expansion in vitro (REM) as described herein.Preferably, human recombinant IL-2 or another suitable IL-2 preparationis added in low concentrations at 3-5 day intervals (typically on “day0”(i.e. at culture initiation) or “day 1”(the day followinginititiation), again on day 5 or 6, and again on day 8 or 9). REMprotocols result in a rapid expansion of T cells, typically in the rangeof a 500- to 3000-fold expansion within 8 to 14 days. Such methods canthus achieve expansion rates that are approximately 100- to 1000-foldmore efficient for each stimulation cycle than previously-describedmethods of culturing human T cells.

Furthermore, REM protocols are applicable to the rapid expansion of anyT cell sub-population including helper T cells and cytolytic T cells;and to T cell clones of many different antigenic specificities (e.g., tocytolytic or helper T cells specific for CMV, HIV, or other viral,bacterial, or tumor-derived antigens). In addition, REM protocols can beused for both small scale growth (e.g. to rapidly expand T cells from10⁴ to 10⁷ cells); or for large-scale expansions (e.g. to rapidly expandT cells from 10⁶ to greater than 10¹⁰ cells); depending on the size ofculture vessel chosen.

REM protocols thus make it possible to efficiently expand T cell clonesfor use in adoptive immunotherapies by dramatically shortening the timerequired to grow the numbers of cells required to restore, enhance, ormodulate human immunity. In the study by Riddell et al. (Science,257:238-240, 1992), once T cell clones were isolated it was necessary toculture the clones for twelve weeks and to pool multiple clones toachieve the highest administered cell dose of 1×10⁹ CD8+ CMV-specific Tcells/m² body surface area. Using REM protocols, the expansion ofindividual T cell clones to greater than 10⁹ cells can be accomplishedin less than three weeks.

With respect to the rapid expansion methods (i.e. “REM” technology), thefollowing abbreviations are used to distinguish the various REMprotocols referred to herein. The basic Riddell protocol (as describedabove and in the cited Riddell patent application), which uses adisproportionately large number of PBMC feeder cells (and preferablyalso EBV-LCL feeder cells) is referred to as “high-PBMC REM” or simply“hp-REM”. Conversely, the methods of the present invention, which do notemploy such large excesses of PBMC feeder cells (and preferably noEBV-LCL feeder cells) are referred to as “low-PBMC REM” or“modified-REM”. Such methods are described in detail below.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: ALaboratory Manual, Second Edition (1989); Animal Cell Culture (R. I.Freshney, Ed., 1987); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos eds. 1987); Handbook of Experimental Immunology,(D. M. Weir and C. C. Blackwell, Eds.); Current Protocols in MolecularBiology (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.Siedman, J. A. Smith, and K. Struhl, eds., 1987); Current Protocols inImmunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach and W. Strober, eds., 1991); Oligonucleotide Synthesis (M. J.Gait Ed., 1984); and the series Methods in Enzymology (Academic Press,Inc.).

All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated herein by reference.

As an aid in understanding this invention, the following is a list ofsome abbreviations commonly used herein:

CTL cytotoxic T lymphocyte(s)

APC antigen-presenting cell(s)

CMV cytomegalovirus

HIV human immunodeficiency virus

EBV Epstein Barr virus

hIL-2 human interleukin-2

MHC major histocompatibility complex

PBMC peripheral blood mononuclear cell(s)

EBV-LCL EBV-transformed lymphoblastoid cell line (sometimes abbreviatedas simply “LCL”)

PBS phosphate buffered solution

REM rapid expansion method

hp-REM high-PBMC REM

lp-REM low-PBMC or “modified” REM

A “cytokine,” as used herein, refers to any of a variety ofintercellular signaling molecules (the best known of which are involvedin the regulation of mammalian somatic cells). A number of families ofcytokines, both growth promoting and growth inhibitory in their effects,have been characterized including, for example: interleukins (such asIL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 (P40),IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15); CSF-type cytokines suchas GM-CSF, G-CSF, M-CSF, LIF, EPO, TPO (“thrombopoietin”), TNF-α, andTNF-β); interferons (such as IFN-α, IFN-β, IFN-γ); cytokines of theTGF-β family (such as TGF-β1, TGF-β2, TGF-β3, inhibin A, inhibin B,activin A, activin B); growth factors (such as EGF, VEGF, SCF (“stemcell factor” or “steel factor”), TGF-α, aFGF, bFGF, KGF, PDGF-A, PDGF-B,PD-ECGF, INS, IGF-I, IGF-II, NGF-β); α-type intercrine cytokines (suchas IL-8, GRO/MGSA, PF-4, PBP/CTAP/βTG, IP-10, MIP-2, KC, 9E3); andβ-type intercrine cytokines (such as MCAF, ACT-2/PAT 744/G26, LD-78/PAT464, RANTES, G26, I309, JE, TCA3, MIP-1α, B, CRG-2); and chemotacticfactors (such as NAP-1, MCP-1, MIP-1α, MIP-1β, MIP-2, SISβ, SISδ, SISε,PF-4, PBP, γIP-10, MGSA). A number of other cytokines are also known tothose of skill in the art. The sources, characteristics, targets andeffector activities of these cytokines have been described and, for manyof the cytokines, the DNA sequences encoding the molecules are alsoknown; see, e.g., R. Callard & A. Gearing, The Cytokine Facts Book(Academic Press, 1994), and the particular publications reviewed and/orcited therein, which are hereby incorporated by reference in theirentirety. As referenced in catalogs such as The Cytokine Facts Book,many of the DNA and/or protein sequences encoding such cytokines arealso generally available from sequence databases such as GENBANK (DNA);and/or SWISSPROT (protein). Typically, cloned DNA encoding suchcytokines will already be available as plasmids, although it is alsopossible to synthesize polynucleotides encoding the cytokines based uponthe published sequence information. Polynucleotides encoding thecytokines can also be obtained using polymerase chain reaction (PCR)methodology, as described in the art. See, e.g., Mullis & Faloona, Met.Enzymology, 155: 355 (1987). The detection, purification, andcharacterization of cytokines, including assays for identifying newcytokines effective upon a given cell type, have also been described ina number of publications as well as the references referred to herein.See, e.g., Lymphokines and Interferons, 1987; and DeMaeyer, E., et al.,“Interferons and Other Regulatory Cytokines,” (John Wiley & Sons 1988).

A mammalian “cell line”, as used herein, refers to a population ofmammalian cells (preferably human cells) that have undergone repeatedpropagation in vitro; as distinguished from “primary cells” taken froman individual such as a human. Generally, a mammalian cell line willhave been propagated in vitro for at least about 10 generations, moretypically at least about 40 generations, most typically at least about100 generations. Most preferably, the mammalian cell line can bepropagated and maintained long term (i.e., at least several months invitro, preferably at least a year). Such cell lines would include, butare not limited to, “clonal” lines (in which all of cells of thepopulation are derived from a single ancestral cell). Conversely, amixed peripheral blood population such as PBMCs would not constitute amammalian cell line. A mammalian cell line for use in the presentinvention may, however, contain a cell type found in peripheral bloodbut in that case the cell type will generally be present at a frequencymuch higher than is normally found in human peripheral blood mononuclearcells (at least twice the frequency generally found in human peripheralblood mononuclear cells; preferably at least five times, at least tentimes, at least twenty times or at least fifty times the frequencygenerally found in human peripheral blood mononuclear cells). Aparticular “cell type” might be, for example, one of the cell typestypically found in peripheral blood (such as B lymphocytes, monocytes,cytotoxic T lymphocytes, helper T lymphocytes, granulocytes, eosinophilsor NK cells); or of a cell type not normally found in peripheral blood(such as fibroblasts, endothelial cells, etc.); or a more specificsubpopulation of such a cell type (e.g. a subpopulation that isrelatively homogeneous with respect to antigen-specificity or expressionof a particular receptor). Thus, a cell line might be relativelyhomogeneous with respect to attributes such as antigen-specificity orcell surface receptors/ligands, as discussed in more detail below. Byway of illustration, a receptor-specific monocyte line refers to apopulation of cells in vitro in which the majority of cells aremonocytes possessing a particular cell surface receptor (which cell linemight have been obtained for example by transforming a population ofmonocytes with genes expressing the particular receptor). Again, by wayof illustration, an antigen-specific CTL cell line refers to apopulation of cells in vitro in which the majority of cells arecytotoxic T lymphocytes specific for a particular antigen such as aviral, bacterial or tumor antigen (which cell line might have beenobtained for example by exposing a population of T cells to repeatedstimulation with a particular antigen and subsequently enriching forantigen-specific CTLs).

Preferably, such a cell line for use with the present invention will berendered non-dividing prior to use in the modified-REM culture (e.g., byirradiation). However, one can alternatively (or in addition) employ acell line that is dividing (preferably at a rate similar to or slowerthan the expanding T cells) but which can be subsequently eliminated byvirtue of its having a negative selectable marker (e.g., a suicide genethat can be used to inhibit or kill cells carrying the gene, or a cellsurface marker that can be used to isolate and/or eliminate cellscarrying the marker). In the latter case, the cell line can be allowedto expand to some degree in the REM culture before being negativelyselected.

Preferably, mammalian cell lines to be used with the present inventionare relatively homogeneous lines (i.e. at least 50% of the cells are ofa particular cell type, more preferably at least 70%, at least 90%, atleast 95% or at least 99% of the cells are of a particular cell type).It should be noted, however, that T cells to be expanded by exposure tosuch a cell line might also be exposed to additional cell lines (at thesame time or in sequence). Thus, by way of illustration, a modified-REMculture (containing a T lymphocyte population to be expanded) might beexposed to one mammalian cell line or to several such lines. Formodified-REM, T cells to be expanded will be exposed to at least onesuch mammalian cell line and/or to a non-cellular mixture of factors(including, e.g., cytokines, antibodies, soluble ligands, etc.), asdiscussed herein.

The T cells to be propagated in culture (i.e., the “target” T-cells) canbe obtained from the subject to be treated. Alternatively, T cells canbe obtained from a source other than the subject to be treated, in whichcase the recipient and transferred cells are preferably immunologicallycompatible (or the receipient is otherwise made immuno-tolerant of thetransferred cells). Typically, the target T cells are derived fromtissue, bone marrow, fetal tissue, or peripheral blood. Preferably, thecells are derived from peripheral blood. If the T cells are derived fromtissues, single cell suspensions can be prepared using a suitable mediumor diluent.

Mononuclear cells containing the T lymphocytes can be isolated from theheterogenous population according to any of the methods well known inthe art. As illustrative examples, Ficoll-Hypaque gradientcentrifugation, fluorescence-activated cell sorting (FACs), panning onmonoclonal antibody coated plates, and/or magnetic separation techniquescan be used (separately or in combination) to obtain purifiedpopulations of cells for expansion according to the present invention.Antigen-specific T cells can be isolated by standard culture techniquesknown in the art involving initial activation of antigen-specific T cellprecursors by stimulation with antigen-presenting cells and, for aclonal population, by limiting dilution cultures using techniques knownin the art, such as those described in Riddell and Greenberg (J.Immunol. Meth., 128:189-201, 1990); and Riddell et al. (J.Immunol.,146:2795-2804, 1991). See also, the Examples below. T cell clonesisolated in microwells in limiting dilution cultures typically haveexpanded from a single cell to 2×10⁴ to 5×10⁵ cells after 14 days.

For expansion, T cells can be placed in appropriate culture media inplastic culture vessels with T cell stimulatory components as describedherein. The initial phase of rapid expansion is generally carried out ina culture vessel, the size of which depends upon the number of targetcells, and which may typically be a 25 cm² flask. The size of theculture vessel used for subsequent cycles of T cell expansion depends onthe starting number of T cells and the number of cells needed. Typicalstarting cell numbers for different sized culture vessels are asfollows: 5×10⁴ to 2×10⁵—approximately 25cm² flask; 2×10⁵ to5×10⁵—approximately 75² cm flask; 5×10⁵ to 1×10⁶—approximately 225-cm²flask; and 1×10 ⁶ to 2×10⁶—roller bottle. The approximate initial volumeof media used with each flask is: 25 cm²—20-30 ml; 75 cm²—60-90 ml; 225cm²—100-200 ml; roller bottle—500 ml.

For even larger-scale expansions, a variety of culture means can beused, including for example, spinner flasks, cell culture bags, andbioreactors (such as hollow-fiber bioreactors).

As used herein, “feeder cells” are accessory cells that provideco-stimulating functions in conjunction with T cell receptor activation(which can be achieved by ligation of the T cell receptor complex withanti-CD3 monoclonal antibody). PBMC feeder cells for use in REM can beobtained by techniques known in the art, for example by leukaphoresis,which is a standard medical procedure with minimal risks (see, e.g.,Weaver et al., Blood 82:1981-1984, 1993); and these feeder cells can bestored by cryopreservation in liquid nitrogen until use. LCL can begenerated from peripheral blood B cells by transformation with EBV, forexample the B95-8 strain of EBV, using standard methods (see, e.g.,Crossland et al., J. Immunol. 146:4414-20, 1991), or by spontaneousoutgrowth in the presence of cyclosporin A. Such LCL cells will growrapidly and indefinitely in culture.

Prior to adding any feeder cells to the culture vessel (whether PBMCs orcells derived from a cell line as described herein), such feeder cellsare preferably prevented from undergoing mitosis. Techniques forpreventing mitosis are well known in the art and include, for exampleirradiation. For example, any PBMCs can be irradiated with gamma rays inthe range of about 3000 to 4000 rads (preferably PBMCs are irradiated atabout 3600 rads); any LCL can be irradiated with gamma rays in the rangeof about 6000-12,000 rads (preferably LCL are irradiated at about 10,000rads); and any cells derived from other cell lines can also beirradiated with gamma rays in the range of about 6000-12,000 rads. Asdiscussed above, negatively selectable feeder cells can also be used.

Since the antigen specificity of the T cell clone is generally definedprior to expanding the cell in the culture system, either autologous orallogeneic feeder cells can be used to support T cell growth. Theability to use allogeneic feeder cells is important in situations inwhich the patient is infected with a virus that is present in PBMC,e.g., HIV, that could therefore contaminate the T cell cultures. In suchcircumstances, the use of allogeneic feeder cells derived from anindividual that is screened and deemed to be a suitable blood donor byAmerican Red Cross criteria can be used in the culture method.

The T cell receptor activation signal (normally provided by antigen andantigen-presenting cells) can be achieved by the addition anti-CD3monoclonal antibodies to the culture system. The anti-CD3 monoclonalantibody most commonly used is “OKT3”, which is commercially availablefrom Ortho Pharmaceuticals in a formulation suitable for clinical use.The use of anti-CD3 (“αCD3”) mAb rather than antigen as a means ofligating the T cell receptor bypasses the need to have a source ofantigen-presenting cells, which for virus-specific T cells would requiremaintaining large numbers of suitable autologous cells and infectingthese cells in vitro with high titer virus. A concentration of anti-CD3monoclonal antibody of at least about 0.5 ng/ml, preferably at leastabout 1 ng/ml, more preferably at least about 2 ng/ml, promoted therapid expansion of the T cells such that a 500- to 3000-fold expansioncan be achieved within about 10 to 13 days of growth. Typically, aconcentration of about 10 ng/ml anti-CD3 monoclonal antibody was used.

Of course, as an alternative to anti-CD3 monoclonal antibody, the T cellreceptors can be activated and the cells stimulated by the addition ofantigen-presenting cells, as described in Riddell et al., J. Immunol.146:2795-2904, 1991. Suitable antigen-presenting cells include, forexample, viral infected cells, tumor cells, and cells pulsed with therelevant peptide antigen.

The culture media for use in the methods of the invention can be any ofthe commercially available media, preferably one containing: RPMI, 25 mMHEPES, 25 μM 2-mercaptoethanol, 4 mM L-glutamine, and 11% human ABserum. Fetal calf serum can be substituted for human AB serum.Preferably, after addition of any feeder cells, anti-CD3 monoclonalantibody, and culture media are added to the target T cells, and themixture is allowed to incubate at 37° C. in a 5% CO₂ humidifiedatmosphere under standard cell culture conditions which are well knownin the art. Typically, such conditions may include venting, and additionof CO₂ if necessary (e.g., 5% CO₂, in a humidified incubator).

Preferably, the medium is also supplemented with interleukin-2 (IL-2).Typically recombinant human IL-2 is used, although a functionalequivalent thereof may also be used. Preferably, IL-2 is added on day 1,and is re-added at 3-5 day intervals. Thus, IL-2 was generally added onday 1, on day 5 or 6, and again on day 8 or 9. Expansion can be improvedby using an IL-2 concentration of at least about 5 U/ml, more preferablyat least about 10 U/ml. Generally, a concentration of about 25 U/ml canbe used.

As described in Riddell et al., supra, antigen-specific T cells expandedusing REM retained their antigen-specific functionality. For example,four different HIV-specific CD8+ cytotoxic T cell clones retained theability to kill virus-infected cells expressing the relevant antigen(i.e. HIV), and did not acquire non-specific cytolytic activitiesagainst irrelevant virus-infected or transformed target cells.Similarly, four different CMV-specific CD8+ cytotoxic T cell clonesretained the ability to kill CMV-infected cells, and did not acquirenon-specific cytolytic activities against irrelevant virus-infected ortransformed target cells. These characteristics were also applicable toCD4+ helper T cells. Thus, antigen-specific CD4+ T cells propagatedusing REM retained the ability to proliferate in response to theappropriate viral antigens and appropriate antigen-presenting cells(APC). Furthermore, antigen-specific T cells cultured under REM werealso capable of entering a quiescent, non-dividing phase of the cellcycle; and were capable of remaining viable for at least 4 weeks invitro. Thus, aliquots of T cells can be removed from the cultures at theend of a stimulation cycle (generally day 12-14), and placed in aculture vessel with a roughly equal number of irradiated PBMC (withoutanti-CD3 mAb, antigen or IL-2).

The addition of irradiated PBMC as feeder cells during storage ofexpanded populations improved the ability of the T cells to enter aresting phase and to remain viable. Preferably, the ratio of PBMC feedercells to resting T cells during storage is at least about 2:1. Withoutthe addition of PBMC feeder cells, viability of the T cells generallydrops significantly (typically to levels of about 10% or less).

As described in Riddell et al., supra, T cells expanded by REM assumed asmall round morphology and 60-95% remained viable by trypan blue dyeexclusion even after 28 days in culture. T cells propagated by hp-REMalso entered a resting phase upon IL-2 withdrawal; and they did notundergo programmed cell death (i.e. apoptosis) upon restimulation viathe antigen-specific T cell receptor. Upon restimulation (e.g. withanti-CD3 mAb or antigen), the T cells required responsiveness to IL-2,and can enter the S and G2 phases of the cell cycle and increased incell number. Such characteristics are believed to be important for invivo survival of the cells and for the efficacy of cellularimmunotherapy. In contrast, certain previously-described methods for thepropagation of T cells have been reported to cause apoptotic cell deathin a proportion of cells after cytokine withdrawal or T cell receptorrestimulation (see, e.g., Boehme S A and Lenardo M J, Eur. J. Immunol.,23:1552-1560, 1992).

There are a number of different circumstances in which the introductionof functional genes into T cells to be used in immunotherapy may bedesirable. For example, the introduced gene or genes may improve theefficacy of therapy by promoting the viability and/or function oftransferred T cells; or they may provide a genetic marker to permitselection and/or evaluation of in vivo survival or migration; or theymay incorporate functions that improve the safety of immunotherapy, forexample, by making the cell susceptible to negative selection in vivo asdescribed by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); andRiddell et al., Human Gene Therapy 3:319-338 (1992); see also thepublications of WO/92 08796 and WO/94 28143 by Lupton et al., describingthe use of bifunctional selectable fusion genes derived from fusing adominant positive selectable marker with a negative selectable marker.

Various infection techniques have been developed which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a currently preferred approach to the transduction of Tlymphocytes of the present invention. The viral vectors which have beenused in this way include virus vectors derived from simian virus 40(SV40) (see, e.g., Karlsson et al., Proc. Natl. Acad. Sci. USA 8482:158, 1985); adenoviruses (see, e.g., Karlsson et al., EMBO J. 5:2377,1986); adeno-associated virus (AAV) (see, e.g., B. J. Carter, CurrentOpinion in Biotechnology 1992, 3:533-539); and retroviruses (see, e.g.,Coffin, 1985, pp. 17-71 in Weiss et al. (eds.), RNA Tumor Viruses, 2nded., Vol. 2, Cold Spring Harbor Laboratory, New York). Thus, genetransfer and expression methods are numerous but essentially function tointroduce and express genetic material in mammalian cells. A number ofthe above techniques have been used to transduce hematopoietic orlymphoid cells, including calcium phosphate transfection (see, e.g.,Berman et al., supra, 1984); protoplast fusion (see, e.g., Deans et al.,supra, 1984); electroporation (see, e.g., Cann et al., Oncogene 3:123,1988); and infection with recombinant adenovirus (see, e.g., Karlsson etal., supra; Reuther et al., Mol. Cell. Biol. 6:123, 1986);adeno-associated virus (see, e.g., LaFace et al., supra); and retrovirusvectors (see e.g., Overell et al., Oncogene 4:1425, 1989). Primary Tlymphocytes have been successfully transduced by electroporation (see,e.g., Cann et al., supra, 1988) and by retroviral infection (see e.g.,Nishihara et al., Cancer Res. 48:4730, 1988; Kasid et al., supra, 1990;and Riddell, S. et al., Human Gene Therapy 3:319-338, 1992).

Retroviral vectors provide a highly efficient method for gene transferinto eukaryotic cells. Moreover, retroviral integration takes place in acontrolled fashion and results in the stable integration of one or a fewcopies of the new genetic information per cell.

Retroviruses are a class of viruses which replicate using avirus-encoded, RNA-directed DNA polymerase, or reverse transcriptase, toreplicate a viral RNA genome to provide a double-stranded DNAintermediate which is incorporated into chromosomal DNA of an avian ormammalian host cell. Most retroviral vectors are derived from murineretroviruses. Retroviruses adaptable for use in accordance with thepresent invention can, however, be derived from any avian or mammaliancell source. These retroviruses are preferably amphotropic, meaning thatthey are capable of infecting host cells of several species, includinghumans. A characteristic feature of retroviral genomes (and retroviralvectors used as described herein) is the retroviral long terminalrepeat, or LTR, which is an untranslated region of about 600 base pairsfound in slightly variant forms at the 5′ and 3′ ends of the retroviralgenome. When incorporated into DNA as a provirus, the retroviral LTRincludes a short direct repeat sequence at each end and signals forinitiation of transcription by RNA polymerase II and 3′ cleavage andpolyadenylation of RNA transcripts. The LTR contains all othercis-acting sequences necessary for viral replication.

A “provirus” refers to the DNA reverse transcript of a retrovirus whichis stably integrated into chromosomal DNA in a suitable host cell, or acloned copy thereof, or a cloned copy of unintegrated intermediate formsof retroviral DNA. Forward transcription of the provirus and assemblyinto infectious virus occurs in the presence of an appropriate helpervirus or in a cell line containing appropriate sequences enablingencapsidation without coincident production of a contaminating helpervirus. Mann et al. (Cell 33:153, 1983) describe the development of celllines (e.g., Ψ2) which can be used to produce helper-free stocks ofrecombinant retrovirus. These cells lines contain integrated retroviralgenomes which lack sequences required in cis for encapsidation, butwhich provide all necessary gene product in trans to produce intactvirions. The RNA transcribed from the integrated mutant provirus cannotitself be packaged, but these cells can encapsidate RNA transcribed froma recombinant retrovirus introduced into the same cell. The resultingvirus particles are infectious, but replication-defective, renderingthem useful vectors which are unable to produce infectious virusfollowing introduction into a cell lacking the complementary geneticinformation enabling encapsidation. Encapsidation in a cell lineharboring trans-acting elements encoding an ecotropic viral envelope(e.g., Ψ2) provides ecotropic (limited host range) progeny virus.Alternatively, assembly in a cell line containing amphotropic packaginggenes (e.g., PA317, ATCC CRL 9078; Miller and Buttimore, Mol. Cell.Biol. 6:2895, 1986) provides amphitropic (broad host range) progenyvirus. Such packing cell lines provide the necessary retroviral gag, poland env proteins in trans. This strategy results in the production ofretroviral particles which are highly infectious for mammalian cells,while being incapable of further replication after they have integratedinto the genome of the target cell. The product of the env gene isresponsible for the binding of the retrovirus to viral receptors on thesurface of the target cell and therefore determines the host range ofthe retrovirus. The PA 317 cells produce retroviral particles with anamphotropic envelope protein, which can transduce cells of human andother species origin. Other packaging cell lines produce particles withecotropic envelope proteins, which are able to transduce only mouse andrat cells.

Numerous retroviral vector constructs have been used successfully toexpress many foreign genes (see, e.g., Coffin, in Weiss et al. (eds.),RNA Tumor Viruses, 2nd ed., vol. 2 (Cold Spring Harbor Laboratory, NewYork, 1985, pp. 17-71). Retroviral vectors with inserted sequences aregenerally functional, and few sequences that are consistently inhibitoryfor retroviral infection have been identified. Functionalpolyadenylation motifs inhibit retroviral replication by blockingretroviral RNA synthesis, and there is an upper size limit ofapproximately 11 kb of sequence which can be packaged into retroviralparticles (Coffin, supra, 1985); however, the presence of multipleinternal promoters, initially thought to be problematic (Coffin, supra,1985), was found to be well tolerated in several retroviral constructs(Overell et al., Mol. Cell. Biol. 8:1803, 1983).

Retroviral vectors have been used as genetic tags by several groups tofollow the development of murine hematopoietic stem cells which havebeen transduced in vitro with retrovirus vectors and transplanted intorecipient mice (Williams et al., Nature 310:476, 1984; Dick et al., Cell42:71, 1985; Keller et al., Nature 318:149, 1985). These studies havedemonstrated that the infected hematopoietic cells reconstitute thehematopoietic and lymphoid tissue of the recipient animals and that thecells display a normal developmental potential in vivo. The marked cellscan be visualized using any of a number of molecular biologicaltechniques which can demonstrate the presence of the retroviral vectorsequences, most notably Southern analysis and PCR (polymerase chainreaction). The ability to mark cells genetically using retroviralvectors is also useful in clinical settings in which the technique canbe used to track grafts of autologous cells. This approach has alreadybeen used to track TILs (tumor-infiltrating lymphocytes) in patientsgiven TIL therapy for terminal cancer treatment by Rosenberg et al. (N.Engl. J. Med. 323:570, 1990). The transduction of these cells with themarker gene was not associated with in vitro cellular dysfunction (Kasidet al., Proc. Natl. Acad. Sci. USA 87:473, 1990).

Many gene products have been expressed in retroviral vectors. This caneither be achieved by placing the sequences to be expressed under thetranscriptional control of the promoter incorporated in the retroviralLTR, or by placing them under the control of a heterologous promoterinserted between the LTRs. The latter strategy provides a way ofcoexpressing a dominant selectable marker gene in the vector, thusallowing selection of cells which are expressing specific vectorsequences.

It is contemplated that overexpression of a stimulatory factor (forexample, a lymphokine or a cytokine) may be toxic to the treatedindividual. Therefore, it is within the scope of the invention toinclude gene segments that cause the T cells of the invention to besusceptible to negative selection in vivo. By “negative selection” ismeant that the infused cell can be eliminated as a result of a change inthe in vivo condition of the individual. The negative selectablephenotype may result from the insertion of a gene that conferssensitivity to an administered agent, for example, a compound. Negativeselectable genes are known in the art, and include, inter alia thefollowing: the Herpes simplex virus type I thymidine kinase (HSV-I TK)gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovirsensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT)gene, the cellular adenine phosphoribosyltransferase (APRT) gene,bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci.USA. 89:33 (1992)).

In addition, it is useful to include in the T cells a positive markerthat enables the selection of cells of the negative selectable phenotypein vitro. The positive selectable marker may be a gene which, upon beingintroduced into the host cell expresses a dominant phenotype permittingpositive selection of cells carrying the gene. Genes of this type areknown in the art, and include, inter alia, hygromycin-Bphosphotransferase gene (hph) which confers resistance to hygromycin B,the aminoglycoside phosphotransferase gene (neo or aph) from Tn5 whichcodes for resistance to the antibiotic G418, the dihydrofolate reductase(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drugresistance (MDR) gene.

Preferably, the positive selectable marker and the negative selectableelement are linked such that loss of the negative selectable elementnecessarily also is accompanied by loss of the positive selectablemarker. Even more preferably, the positive and negative selectablemarkers are fused so that loss of one obligatorily leads to loss of theother. An example of a fused polynucleotide that yields as an expressionproduct a polypeptide that confers both the desired positive andnegative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See Lupton S. D., et al., Mol. and Cell. Biology11:3374-3378, 1991. In addition, in preferred embodiments, thepolynucleotides of the invention encoding the chimeric receptors are inretroviral vectors containing the fused gene, particularly those thatconfer hygromycin B resistance for positive selection in vitro, andganciclovir sensitivity for negative selection in vivo, for example theHyTK retroviral vector described in Lupton, S. D. et al. (1991), supra.See also the publications of PCT/US91/08442 and PCT/US94/05601, by S. D.Lupton, describing the use of bifunctional selectable fusion genesderived from fusing a dominant positive selectable markers with negativeselectable markers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, neo, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Especially preferred markers are bifunctional selectable fusion geneswherein the positive selectable marker is derived from hph or neo, andthe negative selectable marker is derived from cytosine deaminase or aTK gene.

A variety of methods can be employed for transducing T lymphocytes, asis well known in the art. Typically, one can carry out retroviraltransductions as follows: on day 1 after stimulation using REM asdescribed herein, one can provide the cells with 20-30 units/ml IL-2; onday 1, 2, or 3, one half of the medium can be replaced with retroviralsupernatant prepared according to standard methods and the culturessupplemented with 5 μg/ml polybrene and 20-30 units/ml IL-2; on day 4,the cells are washed and placed in fresh culture medium supplementedwith 20-30 units/ml IL-2; on day 5, the exposure to retrovirus can berepeated; on day 6, the cells can be placed in selective medium(containing, e.g., an antibiotic corresponding to an antibioticresistance gene provided in the retroviral vector) supplemented with 30units/ml IL-2; on day 13, viable cells can be separated from dead cellsusing Ficoll Hypaque density gradient separation and then the viablecells can be subcloned using REM.

Using an antigen-specific CTLs (55E1, an EBV-specific CD8+ clonal line)and a retroviral vector (LAPSN, Clowes et al., 1994, J. Clin. Invest.93:644 (which allowed for monitoring of alkaline phosphatase expressionby flow cytometry)), high transduction frequencies can be achieved whenthe cells are exposed to vector on day 1, 2 or 3 after initiation ofREM.

As described above, T cells prepared according to the invention can beused to restore, enhance, and/or modulate immunity in recipientindividuals. By “immunity” is meant a lessening of one or more physicalsymptoms associated with a response to infection by a pathogen, or to atumor, to which the lymphocyte response is directed. The amount of cellsadministered is usually in the range present in normal individuals withimmunity to the pathogen. Thus, CD8+ CD4− cells are usually administeredby infusion, with each infusion in a range of at least 10⁶ to 10¹⁰cells/m², preferably in the range of at least 10⁷ to 10⁹ cells/m². Theclones may be administered by a single infusion, or by multipleinfusions over a range of time. However, since different individuals areexpected to vary in responsiveness, the type and amount of cellsinfused, as well as the number of infusions and the time range overwhich multiple infusions are given are determined by the attendingphysician, and can be determined by routine examination. The generationof sufficient levels of T lymphocytes (including cytotoxic T lymphocytesand/or helper T lymphocytes) is readily achievable using the rapidexpansion method of the present invention, as exemplified herein.

It has also been observed that T cells expanded using REM exhibited veryhigh levels of transduction using vectors such as retroviral vectorswhich will be of great use in the contexts of cellular immunotherapy andgene therapy using lymphocytes.

The examples in Riddell et al., supra, exemplify the basic REM protocol(i.e. hp-REM), and also help to illustrate the general applications ofREM technology to the preparation and use of expanded T cell populationsand, in that regard, exemplify techniques and principles that can alsobe applied in the context of modified-REM.

The examples below illustrate exemplary modifications of the REMtechnology according to the present invention (i.e. modified-REM), toenable a reduction or elimination of the PBMC and/or EBV-LCL feedercells that are characteristic of the hp-REM protocol.

All of the examples presented below are provided as a further guide tothe practitioner of ordinary skill in the art, and are not to beconstrued as limiting the invention in any way.

EXAMPLES Example 1

The Contribution of Monocyte Fc-γ Receptors in Rapid Expansion

The PBMC feeder cells, which are used in large excess to drive hp-REM,are a heterogeneous population of cells including B lymphocytes, Tlymphocytes, monocytes, macrophages, and granulocytes, and NaturalKiller (“NK”) cells.

One of the activities believed to be supplied by PBMCs in the hp-REMprotocol is the provision of Fc-γ receptors which can bind to the Fcportion of IgG antibody molecules. In particular, it is believed that Tcell activation in the hp-REM protocol can be mediated by binding ofFc-γ receptors (“FcγR”) on monocytes within the PBMC population to theFc portion of anti-CD3 antibody (e.g. OKT3), which can thereby be“presented” by the monocytes to T cells within the population to beexpanded. Following such activation, T cells are believed to be capableof initiating an “autocrine” growth stimulatory cycle in which activatedT cells both secrete growth-stimulatory cytokines and also increase theexpression of cell surface receptors for such cytokines. Supplying Fc-γreceptors, or otherwise effectively presenting anti-CD3 antibody, isthus believed to function in the initiation and promotion of T cellexpansion.

Confirming and quantifying the contribution of monocyte Fc-γ receptorsin the hp-REM protocol can be accomplished by depleting monocytes fromthe PBMC feeder cells.

Peripheral blood mononuclear cells can be obtained from any of a varietyof sources, as described above. For the following examples, buffy coatlayers (derived from healthy human donors) were obtained from a RedCross blood bank. PBMCs were isolated using a Ficoll gradient, washedand stored in cell culture media (at 4 degrees Celsius) using standardtechniques as referred to above.

A variety of techniques can be used for separating out various celltypes from a mixed population such as PBMCs. By way of illustration,depletion of the monocyte/macrophage population was performed usingsephadex G-10 chromatography (see, e.g., Section 3.6 in “CurrentProtocols in Immunology” (Wiley Interscience, 1992)). Monocyte depletionwas monitored by flow cytometry following staining with FITC-conjugatedanti-CD14 monoclonal antibody (available from, e.g., PharmaGen). CD14expression before depletion was about 7.4% of total cells. Followingdepletion, it was about 1.5%.

In order to assess the impact of depleting monocytes on the ability ofPBMCs to promote rapid expansion, a standard hp-REM protocol was usedand monocyte-depleted PBMCs were compared with non-depleted PBMCs.

For purposes of illustration, a CTL line (designated “27EB”) wasprepared by procedures analogous to those described above. Briefly,PBMCs were obtained from an individual blood sample and were culturedwith EBV-LCL derived from the same individual. After two weeks, CD8+CTLs were isolated by “panning” with a flask coated with anti-CD8antibodies (e.g. the AIS-CD8+ “CELLector flask” from Applied ImmuneSciences).

For all of these illustrative examples, PBMCs were gamma-irradiated at3600 rads (using a Cs-137 source) and EBV-LCL were irradiated at 10,000rads.

Cultures were generally maintained as described above for hp-REM exceptthat 10% fetal calf serum was used in place of human serum; and IL-2 wasused at 25 units/ml and was generally first added on “day 0” (as opposedto 1 day after culture initiation), and then at 3-5 day intervals(generally, on day 5, and then again on day 8), as otherwise describedabove for the hp-REM protocol. OKT3 was generally used at about 10ng/ml. Cells were typically harvested and quantified after 14 days ofculture.

For this example, cultures were established with 5×10⁴ CTL (“27EB”, asdescribed above), 5×10⁶ irradiated EBV-LCL (i.e. a 100:1 excess overCTLs) (prepared and irradiated as described above), 10 ng/ml OKT3, 25U/ml IL-2 and 2.5×10⁷ irradiated PBMC (i.e. a 500:1 excess over CTLs)(either monocyte-depleted or nondepleted, prepared and irradiated asdescribed above). Typical control cultures would include, for example,cultures without any added CTL. After 14 days of culture, cells wereharvested and quantified.

In the case of the standard hp-REM protocol using nondepleted PBMCs,approximately 4.86×10⁷ T cells were recovered, representing an expansionof approximately 882-fold.

As shown in TABLE 1, when monocyte-depleted PBMCs were used instead,only about 2.7×10⁷ T cells were recovered, indicating that the expansionrate had dropped to about 55% of the control rate.

TABLE 1 Method of T Cell % Control T cell PBMC Cells depletion RecoveryExpansion Nondepleted PBMC none 4.86 × 10⁷ 100% Monocyte-depletedSephadex column 2.70 × 10⁷  55% PBMC

The above results provided further indication that monocytes within thePBMC population apparently contribute significantly to the ability ofPBMCs to bring about the rapid expansion of T cells. In the followingexample, the ability to provide FcγR activity or its equivalent from asource other than PBMCs as a means for reducing the dependence of REM onlarge excesses of PBMC feeder cells.

Example 2

Replacement of Monocyte Fc-γ Receptor Activity in Modified-REM

As discussed above, Fc-γ receptors found on monocytes are believed to beresponsible for a significant portion of the stimulatory activitysupplied by PBMCs in the presence of antibodies such as anti-CD3antibody (e.g. OKT3).

Having identified a stimulatory component supplied by the heterogeneousPBMC population, it is possible to reduce the dependence on PBMCsthemselves by providing that activity (or its equivalent) from anothersource. Preferably, for use in modified-REM as described herein, theidentified stimulatory activity will be provided by a mammalian cellline or as a non-cellular additive to the REM culture. Illustrativeexamples of both are provided below.

a. Use of Mammalian Cell Lines in Modified-REM

Cell lines expressing one of more identified T cell stimulatoryactivities provided by PBMCs (or LCL) can be effectively used to reducethe dependence of REM on such PBMC (or LCL) feeder cells. Where such acell line is to be incorporated into the protocol, it is preferable, asdescribed above, that the cell line not be a potential source ofadventitious agents such as viruses. Accordingly, the supplemental cellline used is preferably not an EBV-transformed cell line (such asEBV-LCL). Also, for the rapid expansion of human T cells, it isgenerally preferable to use a cell line derived from a higher mammal,especially a primate, most preferably a human.

Mammalian cell lines expressing Fc-γ receptors have been described inthe literature and can be obtained from a variety of sources. Forexample, a number of human tumor lines have been demonstrated to expressFc-γ receptors (see, e.g., R. J. Looney et al., J.Immunol.136:1641(1986) (describing K562, an erythroleukemia cell line); S. J. Collins atel. 1977. Nature 270:347 (1977) (describing HL60, a promyelocytic cellline); C. Lozzio and B. Lozzio, Blood 45:321 (1975) (describing U937, ahistiocytic lymphoma cell line); and G. R. Crabtree et al., Cancer Res.38:4268 (1979).

Cell lines expressing Fc-γ receptors can also be readily prepared usingstandard molecular biological techniques. By way of illustration,FcγR-positive cell lines can be obtained by immortalizing cells thatalready express Fc-γ receptors using any of a variety of well-knowntechniques for transforming mammalian cells. Alternatively, an existingcell line such as a human cell line can be genetically modified toexpress Fc-γ receptors by introducing genes encoding Fc-γ receptors intothe cells. Thus, a cell line of choice, such as a human cell linealready expressing a stimulatory component such as a cytokine or a celladhesion-accessory molecule (both of which are discussed below), can befurther modified by introduction of genes encoding an FcγR.

By way of illustration, human monocytes apparently express two distinctFc-γ receptor types (“FcγRI” and “FcγRII”) which differ in theiraffinity for IgG antibody binding, (see, e.g., Ravetch and Kinet Annu.Review of Immunol. 9:457-492, 1991). In particular, FcγRI is generally ahigh affinity receptor (K_(a)=10⁸−10⁹) while FcγRII is generally a loweraffinity receptor (K_(a)=10⁷). The genes for both FcγRI and FcγRII havebeen identified and cloned. Previous studies have demonstrated thatfibroblasts expressing the FcγRII receptor following gene transfer couldeffectively restore anti-CD3-dependent proliferation ofmonocyte-depleted T lymphocyte cultures (see, e.g., Peltz et al., J.Immunol. 141:1891 (1988)). Thus, a cell line genetically modified toexpress FcγR should be capable of supplying a significant portion of thestimulatory activity supplied by PBMCs in the hp-REM protocol. Use ofsuch a cell line, potentially in conjunction with other components suchas cytokines or adhesion-accessory molecules as described below, wouldthereby enable a decrease in the number of PBMCs required for rapidexpansion.

b. Use of Non-cellular Additives for Modified-REM

In addition to providing T cell stimulatory components by way of a cellline, such as described above, it will be possible to provide a numberof components (or their functional equivalents) as non-cellularadditives to the modified-REM culture medium. Thus, as a differentalternative to the FcγR activity apparently contributed by PBMCmonocytes, it will be possible to provide a substitute or structuralequivalent for FcγR activity. For example, an alternative means forachieving “presentation” of antibodies such as anti-CD3 antibodies to Tcells is to conjugate such antibodies to beads (such as sephadex beadsor magnetic beads).

In order to assess the ability of such bead-conjugated antibodies tosubstitute for soluble antibodies (which are presumably presented viaFcγR), we conducted experiments to determine whether anti-CD3-conjugatedmagnetic beads can effectively replace soluble anti-CD3 monoclonalantibodies in the REM protocol.

Anti-CD3-conjugated magnetic beads (“BioMag anti-CD3”) were obtainedfrom Perceptive Diagnostics. The particles used were approximately 1 μmin size and had covalently attached anti-CD3 monoclonal antibodiesloaded at approximately 20 μg antibody/1×10⁷ beads. A range of beads waschosen to approximate the number of antigen presenting cells (“APCs”)estimated to be present within the PBMC population used in hp-REM.

For quantifying the actual impact on hp-REM, T cell expansion culturescontaining 5×10⁴ CTL, 5×10⁶ irradiated EBV-LCL, 2.5×10⁷ irradiatedallogeneic PBMC and 25 U/ml IL-2 were established, essentially asdescribed above in Example 1. Either 10 ng/ml of soluble anti-CD3antibody (OKT3) or various quantities of anti-CD3-conjugated beads wereadded as the T cell activation reagent. Cell cultures were expanded andT cells counted, essentially as described for Example 1.

The results are shown in TABLE 2. While T cell expansion usinganti-CD3-conjugated beads was somewhat less than with soluble OKT3 (inthe range of about 80%), the results suggest that antibody-coated beadswould be capable of inducing substantial levels of T cellactivation/proliferation within a modified REM protocol. More detailedquantification of the relative role of anti-CD3 presentation as comparedto other activities potentially provided by APCs within the PBMCpopulation can be readily obtained by assessing the expansion ratesobtainable with anti-CD3 beads using APC-depleted cultures, either inthe presence or absence of various cytokines or other solublestimulatory factors (which are described in more detail below).

TABLE 2 Anti-CD3 source T Cell Recovery T Cell Expansion Solubleanti-CD3 Ab (OKT3) 6.52 × 10⁷ 1304-fold   1 × 10⁷ BioMag anti-CD3 5.36 ×10⁷ 1071-fold   5 × 10⁶ BioMag anti-CD3  5.0 × 10⁷ 1000-fold 2.5 × 10⁶BioMag anti-CD3 5.25 × 10⁷ 1051-fold

Comparisons of the relative efficiency of providing various PBMCreplacement components, as described herein, can be readily achieved bystandard titration analyses in which the various components are addedback at varying concentrations to a PBMC-limited REM culture (i.e. aculture in which PBMCs are included at a sub-optimal level). By way ofillustration, experiments described below assessed the impact of addingvarious combinations of exogenous cytokines to sub-optimized hp-REMcultures in which the PBMC population had been reduced to one-half orone-quarter of an optimal starting level. Analogous assays can bereadily performed for other components such as cells expressing FcγR oradhesion-accessory components, anti-CD3-conjugated beads, and/or othersoluble stimulatory factors (such as monoclonal antibodies directed to Tcell surface components), as described in more detail below.

Example 3

The Contribution of B Cells in Rapid Expansion

In order to quantify the contribution of B lymphocytes to the stimulussupplied by PBMCs, we examined the relative ability of B-cell-depletedPBMC populations to support REM.

Isolation of cells such as B cells (or other cells referred to herein)can be conveniently achieved using antibodies directed to a cell surfacemarker known to be present on the cells to be depleted. A variety ofsuch markers are well known, including the various “CD” or “cluster ofdifferentiation” markers; and antibodies to many such markers arereadily obtainable. Also, for many such markers, beads conjugated withthe antibodies are readily available and can greatly facilitate cellseparation.

By way of illustration, CD 19 is a well-known cell surface marker for Blymphocytes. Magnetic beads that had been conjugated with anti-CD19antibodies were obtained from Dynal, and were used to deplete a PBMCpopulation of B cells, following standard procedures as described by themanufacturer.

Depletion was evaluated by fluorescence activated cell sorting (“FACS”)after CD19 staining. The PBMC population was estimated to containapproximately 12% B cells prior to depletion and less than 1% B cellsafter depletion.

For testing the impact of B cell depletion on hp-REM, T cell expansioncultures containing 5×10⁴ CTL, 5×10⁶ irradiated EBV-LCL, 2.5×10⁷irradiated allogeneic PBMC (B-cell-depleted or nondepleted), 10 ng/mlOKT3 and 25 U/ml IL-2 were established; and, after 14 days, T cells wereharvested and quantified as described above. The results, shown in TABLE3, suggested that B cells also contribute to the stimulating activitysupplied by PBMCs.

TABLE 3 Method of T Cell % Control T Cell PBMC Cells depletion RecoveryExpansion Nondepleted PBMC none 4.28 × 10⁷ 100% B-cell-depletedAnti-CD19  9.8 × 10⁷  23% PBMC Magnetic Beads

The decreased levels of T cell expansion in this and the precedingexperiments suggests a role for monocytes and B cells as antigenpresenting cells (“APCs”) in the hp-REM protocol. (The inability toinhibit T cell expansion to the levels observed in Example 1 to thelevels observed in this experiment may be a result of differences incell depletion by the various methods used and/or the presence of smallnumbers of APC undetected by the assays used. It should also be notedthat monocyte depletion as measured in Example 1 only reduced themonocyte population from about 7.5% to about 1.5%.)

There are a number of other well-known techniques that can be used todeplete various cell types from the PBMC population and that cantherefore be used to provide additional confirmation and quantificationof the results described herein. Thus, for example, nylon wool can beused to remove both monocytes and B cells (as well as any fibroblasts)from the PBMC population. The replacement of various APC activities inmodified-REM is further described in the following example.

Example 4

Replacement of Various APC Activities in Modified-REM

The results obtained in the B-cell-depletion and monocyte-depletionexperiments, described above, indicated that putative APCs in the PBMCpopulation appear to contribute to the stimulus supplied by PBMCs. Asdescribed in Examples 1-2, the role of FcγR activity in presentation ofanti-CD3 antibody is expected to account for some portion of theactivity provided by putative APCs. Such FcγR activity can be suppliedby another (non-PBMC) source, e.g. a cell line expressing FcγR oranti-CD3-conjugated beads, as also described above.

It is believed that APCs within the PBMC population also contributeadhesion-accessory molecules and stimulatory cytokines that would beexpected to further enhance the activation/proliferation process.(Furthermore, as described below, T lymphocytes within the PBMCpopulation are also expected to produce stimulatory cytokines as aresult of activation via the anti-CD3 antibody.) The roles of suchadhesion-accessory molecules and cytokines are described in more detailin the examples below.

Example 5

The Contribution of Cytokines in REM

Although anti-CD3 antibody (e.g. OKT3) is used to activate and inducethe proliferation of T cell clones for their in vitro expansion, theγ-irradiated feeder PBMC population also contains a substantialpopulation of T lymphocytes that are believed to be activatable by theanti-CD3 antibody. While such irradiated feeder cells are incapable ofdividing, their activation via anti-CD3 antibody is believed to resultin the secretion of multiple cytokines which can provide additionallympho-proliferative signals. For example, in addition to IL-2, anti-CD3activation of T cells is believed to result in the secretion of otherstimulatory cytokines including IL-1 α and β, IL-6, IL-8, GM-CSF, IFN-αand TNFα and β (du Moulin et. al. 1994. Cytotechnology 15:365). It isbelieved that the secretion of one or more of those cytokines cancontribute substantially to the proliferative stimulus provided by PBMCswithin the hp-REM protocol. Of the numerous other cytokines that havebeen characterized, a number of these are known to stimulate the growthof T cells, including, for example, IL-7 and IL-15. Others can bereadily screened for their ability to enhance T cell proliferation andfor their relative ability to reduce the dependence of REM on largenumbers of PBMCs, as described herein.

By way of illustration, we analyzed the ability of a number ofexogenously-supplied cytokines to reconstitute T cell expansion in REMcultures in which the numbers of PBMC feeder cells had been reduced tosub-optimal levels, in order to quantify the potential role of suchcytokines in promoting REM.

Following procedures essentially analogous to those described above,cultures containing 5×10⁴ CTL, 5×10⁶ EBV-LCL, 10 ng/ml OKT3 and 25 U/mlIL-2 were established with either 2.5×10⁷ irradiated PBMC (100%control), 1.25×10⁷ irradiated PBMC (50%), or 6.12×10⁶ irradiated PBMC(25%).

It is believed that a number of cytokines can act synergistically withIL-2 to promote T cell proliferation. In this illustrative experiment,the following exogenous cytokines were added to the cultures eitheralone or in various combinations as described: IL-1 (40 U/ml), IL-4 (200U/ml), IL-6 (500 U/ml) and IL-12 (20 U/ml).

The results, shown in TABLE 4, confirmed that such cytokines cansubstantially enhance T cell expansion when PBMC populations are reducedto sub-optimal levels. It is not unexpected that expansion levels werenot returned to that observed with the optimal number of PBMC feeders,because the PBMC population is believed to supply additional stimulatoryactivities as described herein. The data suggest that replacement ofIL-4 with IL-12 in a cytokine cocktail may further enhanceproliferation. The properties, sources, and DNA and protein sequences ofmany such cytokines are described in cytokine reference books such as“The Cytokine Facts Book” by R. Callard et al., supra. To take a singleexample for purposes of illustration, IL-12 is known to be aheterodimeric cytokine comprising two peptide chains (p35 and p40) thatinduces IFNγ production by T lymphocytes and co-stimulates theproliferation of peripheral blood lymphocytes. IL-12 also stimulatesproliferation and differentiation of TH1 T lymphocytes, and is known tobe produced by B cells, monocytes/macrophages, and B lymphoblastoidcells. The complete amino acid sequences for both the p35 and p40 chainsare known and available on Genbank (Accession numbers provided inCallard). The IL-12 receptor has also been characterized (id.).

Additional cytokines and cocktails thereof can readily be tested in ananalogous manner; and a comparison of stimulatory cocktails can then bemade using even lower levels of PBMCs.

TABLE 4 Added Cell Percent Cytokine(s) PBMC Recovery Expansion ControlIL-2  2.5 × 10⁷ 4.4 × 10⁷ 882-fold 100%  IL-2 1.25 × 10⁷ 2.8 × 10⁷564-fold 64% IL-2 6.12 × 10⁶ 1.8 × 10⁷ 360-fold 41% IL-2 + IL-1 1.25 ×10⁷ 3.2 × 10⁷ 648-fold 73% IL-2 + IL-1 6.12 × 10⁶ 2.4 × 10⁷ 486-fold 55%IL-2 + IL-4 1.25 × 10⁷ 2.0 × 10⁷ 402-fold 46% IL-2 + IL-4 6.12 × 10⁶ 1.5× 10⁷ 295-fold 33% IL-2 + IL-6 1.25 × 10⁷ 3.2 × 10⁷ 636-fold 72% IL-2 +IL-6 6.12 × 10⁶ 3.0 × 10⁷ 606-fold 69% IL-2 + IL-12 1.25 × 10⁷ 4.65 ×10⁷  930-fold 105%  IL-2 + IL-12 6.12 × 10⁶ 2.88 × 10⁷  558-fold 63%IL-2 + IL-1 + 1.25 × 10⁷ 3.8 × 10⁷ 768-fold 87% IL-4 + IL-6 IL-2 +IL-1 + 6.12 × 10⁶ 3.1 × 10⁷ 618-fold 70% IL-4 + IL-6

Further evidence that soluble components of the feeder cell supernatantcan provide an effective stimulus for low-PBMC REM was obtained byreducing the PBMC population to sub-optimal levels and using a REMsupernatant to provide soluble stimulatory signals.

Briefly, a standard hp-REM protocol was performed as described above,using an antigen-specific CTL clone and performing a 48-hour REMexpansion with PBMC (500:1), EBV-LCL (100:1), anti-CD3 antibody (10ng/ml), and recombinant human IL-2 (25 units/ml). After 48 hours, thecells were harvested and the supernatant (“REM supernatant”) wasexamined as a source of soluble stimulatory factors in a REM expansionin which PBMC were reduced to sub-optimal levels (i.e. ½, ¼ or ⅛ ofoptimal or “SOP”).

The results, as shown in TABLE 5, confirm that such soluble factors canprovide an effective stimulatory signal in the context of low-PBMC REM.In particular, a large proportion of the reduction in fold proliferationlevels observed when PBMC are reduced can be overcome by using the REMsupernatant in place of the standard medium. In addition, the more thePBMC were reduced (i.e. to ⅛ of optimum), the greater was the observedeffect from using the REM supernatant (1022-fold average expansion usingthe REM supernatant versus 359-fold expansion without). Suchsupernatants and/or their components such as individual cytokines or“cocktails” thereof can thus be used to reduce the need for conductingREM with large excesses of feeder cells such as PBMCs).

TABLE 5 Avg. Fold Std. Medium PBMC Proliferation Dev. SOP MEDIUM SOP1255 ±160  1/2 SOP 1178 ±64 1/4 SOP  996 ±23 1/8 SOP  359 ±29 48 HR. REMSUP. SOP 1253 ±144  1/2 SOP 1218 ±73 1/4 SOP 1178 ±89 1/8 SOP 1022 ±77

Example 6

Replacement of Cytokine Activity in Modified-REM

As described above, a large number of cytokines have been described andare widely available, including a number of cytokines that are known tostimulate T lymphocytes. As will be apparent to those of skill in theart, such cytokines (whether or not they were previously known tostimulate T cells) can be readily tested for their ability to augmentrapid expansion using methods such as those above. In addition, for anyof the rapid expansion techniques described herein, the resultingexpanded T cells can be monitored for the maintenance of various desiredcharacteristics, using methods such as those illustrated above forhp-REM.

Cytokines to be used in modified-REM can be introduced to the target Tcells in any of several ways as illustrated herein. Thus, for example,one or more cytokines can be added to the medium, as exemplified above.Alternatively, or in addition, cytokines can also be supplied by cellssecreting the cytokines into the REM medium. Thus, by way ofillustration, a mammalian cell line known to secrete a particularcytokine or combination of cytokines can be used. Alternatively, amammalian cell line that does not already secrete a particular cytokine(or that secretes it at suboptimal levels) can be readily modified byintroducing a gene encoding the desired cytokine. As is well known, thegene can be placed under the control of any of a variety of promoters(as alternatives to its original promoter) so that expression of thecytokine can be controlled to maximize its effectiveness. The entiresequences for a large number of cytokines are known and encoding DNA isoften available. Many such sequences are published in nucleic acidand/or protein databases (such as GenEMBL, GENBANK or Swissprot); see,e.g., the Cytokine Facts Book, R. E. Callard et al., Academic Press,1994). Also, as described above, such additional mammalian cell linescan be modified to provide several T cell stimulatory activities atonce.

Example 7

The Role of Accessory-Adhesion Molecules in Rapid Expansion

As discussed above, APCs such as monocytes and B cells also provideother T cell co-stimulatory signals which serve to enhance T cellactivation/proliferation. Thus, while T cell activation involves thespecific recognition of MHC-bound antigenic peptides on the surface ofAPCs (which interact with the T cell receptor/CD3 complex), a number ofantigen-non-specific receptor:ligand interactions between APCs and Tcells can further enhance T cell activation/proliferation. Inparticular, APCs express ligands for a variety of receptors on T cells,and it appears that T cell activation/proliferation is the result of acombination of signals delivered through the T cell receptor and othersignaling molecules. A number of such receptor:ligand interactions havealready been identified and, for a number of those, inhibition of thereceptor:ligand interactions have been reported to inhibit T cellproliferation and cytokine secretion. By way of illustration, a numberof receptor:ligand pairs that are considered likely to play a role in Tcell activation/proliferation are listed in TABLE 6 below.

TABLE 6 Receptor (T cell) Ligand (APC) CD4 Class II MHC CD8 Class I MHCCD11a (LFA-1) CD54 (ICAM-1) and ICAM 2 & 3 CD2 CD58 (LFA-3) CD5 CD72CD49d (VLA-4) fibronectin (FN) CD27 ligand to CD27 CD28 CD80 (B7.1) andCD86 (B7.2) CD44 hyaluronate

While many of these molecules have been reported to function in adhesion(enhancing cell:cell and/or cell:substrate interactions), many have alsobeen shown to deliver T cell co-stimulatory signals such as enhancingintracellular calcium and the activation of PI and PKC (see, e.g.,Geppert et al. 1990. Immunol. Reviews 117:5-66).

The interactions of such adhesion-accessory molecules as described abovehave been shown to positively enhance activation of resting Tlymphocytes. Antibodies which bind these accessory molecules have beenshown, under specific conditions, to provide T cell activation signals(see, e.g., the references cited below). Also, the addition of purifiedaccessory molecule ligands ICAM-1 and LFA-3 (ligands for CD11a and CD2respectively) to purified T cells being stimulated with anti-CD3monoclonal antibody has been shown to provide co-stimulatory signals forT cell activation and proliferation (see, e.g., Semnani et al. 1994. J.Exp. Med. 180:2125).

Thus, various antibodies directed against CD4 and CD8 are capable ofeither inhibiting T cell activation (see, e.g., I. Bank and L. Chess.1985. J. Exp. Med. 162:1194; G. A. van Seventer. 1986. Eur. J. Immunol.16:1363) or synergizing with anti-CD3 mAb to induce T cell proliferation(see, e.g., F. Emmrich et al. 1986. PNAS 83:8298; T. Owens et al. 1987.PNAS 84:9209; K. Saizawa et al. 1987. Nature 328:260). As is well knownby those of skill in the art, a collection of antibodies raised againsta particular antigen would be expected to contain antibodies binding toa variety of different sites on the antigen.

A number of studies have shown that antibodies to otheradhesion-accessory molecules are capable of augmenting T cellstimulation/proliferation. By way of illustration, see, e.g., J. A.Ledbetter et al. 1985. J. Immunol 135:2331 (antibodies directed to CD5and CD28 augment anti-CD3-induced T cell proliferation); P. J. Martin etal. 1986. J. Immunol. 136:3282 (antibodies to CD28 augmentanti-CD3-induced T cell proliferation); R. Galandrini et al. 1993. J.Immunol. 150:4225, and Y. Shimizu. 1989. J. Immunol 143:2457 (antibodiesdirected against CD44 augment anti-CD3 induced T cell proliferation); S.C. Meur et al. 1984. Cell 36:897 (antibodies directed against the T11.2and T11.3 epitopes of CD2 stimulate T cell proliferation); R. van Lier.1987. J. Immunol. 139:1589 (antibodies directed against CD27 augmentanti-CD3-induced T cell proliferation); Bossy et al. 1995. Eur. J.Immunol. 25:459 (antibodies to CD50 (ICAM-3) augment anti-CD3-induced Tcell proliferation); M. C. Wacholtz et al. 1989. J. Exp. Med. 170:431(antibodies directed to LFA-1 augment anti-CD3-induced proliferationwhen the two antibodies are crosslinked on the T cell surface); G. A.van Seventer et. al. 1990. J. Immunol. 144:4579 (purified ICAM-1immobilized on plastic with anti-CD3 mAb co-stimulates T cellproliferation via the LFA-1 molecule); Y. Shimizu et al. 1990. J.Immunol 145:59 (purified fibronectin on plastic with anti-CD3 mAbco-stimulates T cell proliferation, and antibodies to VLA4 and VLA5inhibited this activity indicating the role of VLA4 and VLA5 asco-stimulatory T cell receptors); N. K. Damle et al. 1992. J. Immunol.148:1985 (soluble ICAM-1, B7-1, LFA-3 and VCAM augment anti-CD3-inducedT cell proliferation). Quantification of the relative contribution ofsuch adhesion-accessory factors within the REM protocol can be readilyaccomplished using deletion techniques and titration experiments inPBMC-limited hp-REM assays analogous to those illustrated above for thecombinations of various cytokines.

Example 8

Replacement of Adhesion-Accessory Molecule Activity in Modified-REM

In an analogous manner to the modifications described above, and perhapsin combination with such modifications, the REM protocol can thus bemodified to include a characterized cell line expressing high levels ofthese receptor ligands (obtained by, e.g., gene modification of a cellline of choice or by the identification of established cell linesalready expressing such molecules). It is also possible to utilizeantibodies directed against accessory molecules known to induce signaltransduction and/or to use purified accessory ligand molecules as meansof substituting for the corresponding activity provided by the PBMCfeeder cells, thereby enabling a reduction in the number of PBMCsrequired to drive REM.

Example 9

Replacement of Additional Stimulatory Activities Provided by EBV-LCL

While EBV-LCL do not appear to be sufficient for achieving maximal Tcell expansion, they are capable of augmenting expansion in the hp-REMprotocol. Analysis of EBV-LCL has indicated that they express adhesionmolecules such as LFA-1, ICAM-1, and LFA-3, as well as FcγR. Inaddition, EBV-LCL secrete IL-1 (Liu et al. Cell. Immunol, 108:64-75,1987) and IL-12 (Kobayashi et al., 1989. J. Exp. Med. 170:827), both ofwhich are also secreted by APCs.

As described above, it is believed that such components can be readilysupplied by other sources—thereby reducing the need for the largenumbers of PBMC and/or EBV-LCL feeder cells characteristic of hp-REM.

Example 10

The use of anti-CD21 Antibody in Modified REM

CD21 is an accessory molecule expressed on mature B lymphocytes and, atlow levels, on T lymphocytes. We examined the ability of a molecule thatbinds to CD21 to provide a stimulatory signal in the context of modifiedREM.

In a first set of experiments, we used plate-bound anti-CD21 antibody toexamine the ability to provide a stimulatory signal in modified REM inwhich the EBV-LCL feeder population was completely eliminated. Twodifferent antigen-specific CTL clones (“R7” which isalloantigen-specific, and “11E2” which is EBV-specific) were tested in amodified REM procedure in which EBV-LCL were eliminated, but othercomponents were maintained as described above (PBMC at 500:1, IL-2 at 25units/ml). Anti-CD21 antibodies are available from commercial sources.We used the anti-CD21 antibody available from Pharmingen. Anti-CD3antibody was also used, and was bound to plates, as with anti-CD21.Cultures were expanded over a two week standard REM cycle, essentiallyas described above.

The data, as shown in TABLE 7, revealed that the inclusion of anti-CD21antibody resulted in a large increase in the fold proliferationobtainable without the use of EBV-LCL (to 650% of control and 408% ofcontrol for R7 and 11E2, respectively).

A second set of experiments, performed using soluble anti-CD21 antibody,provided additional confirmatory data. In particular, a range ofanti-CD21 concentrations was used in REM as above, except that bothanti-CD21 and anti-CD3 were supplied as soluble antibodies (anti-CD21 atconcentrations ranging from 0 ng/ml to 1.75 ng/ml; anti-CD3 at 10ng/ml).

As shown in TABLE 8, the removal of all EBV-LCL feeder cells from thecultures resulted in a substantial reduction in the average foldproliferation (to 10% of control and 14% of control for R7 and 11E2,respectively). The addition of even small amounts of anti-CD21 antibodyto the culture media resulted in a large increase in fold proliferation(to 72% of control and 57% of control for R7 and 11E2, respectively).

While anti-CD21 antibody provides a conveninet method for enhancing thestimulatory signal, it is also possible to stimulate CD21 in other ways.For example, in addition to anti-CD21 antibody, other molecules that canbe used to bind to CD21 include C3d, C3dg, iC3b and gp350/220 of EBV(see, e.g., W. Timens et al., pages 516-518 in “Leucocyte Typing V,White Cell Differentiation Antigens,” Schlossman, S. F., et al. (eds.),Oxford University Press, Oxford, 1995). Also, as described above, whilesuch T cell stimulatory components can be provided as soluble factors inthe modified REM medium, they can also be provided by a cell lineincluded in the medium (e.g., a cell line that secretes or presents amolecule that binds to CD21).

TABLE 7 Fold % Clone Specificity Stimulation Proliferation Control R7Alloantigen anti-CD3  96 100% anti-CD3 + anti-CD21 624 650% 11E2 EBVanti-CD3  48 100% anti-CD3 + anti-CD21 196 408%

TABLE 8 Fold Clone Condition Proliferation % Control R7 SOP REM 900100%  1.75 ng/ml anti-CD21 228 25% 1.25 ng/ml anti-CD21 156 17% 0.625ng/ml anti-CD21 516 57% 0.325 ng/ml anti-CD21 372 41% 0 ng/ml anti-CD21 90 10% 11E2 SOP REM 420 100%  1.75 ng/ml anti-CD21  96 24% 1.25 ng/mlanti-CD21 192 48% 0.625 ng/ml anti-CD21 288 72% 0.325 ng/ml anti-CD21132 33% 0 ng/ml anti-CD21  60 14%

What is claimed is:
 1. A method for expanding an initial T lymphocytepopulation in culture medium in vitro, comprising the steps of: addingan initial T lymphocyte population to a culture medium in vitro; addingto the culture medium a non-dividing mammalian cell line expressing atleast one T-cell-stimulatory component selected from the groupconsisting of an Fc-γ receptor, a cell adhesion-accessory molecule, amolecule that binds CD21, and a cytokine, wherein said cell line is notan EBV-transformed lymphoblastoid cell line (LCL); adding IL-2 to theculture medium, wherein the concentration of IL-2 is at least 10units/ml; and incubating the culture, thereby expanding the T lymphocytepopulation.
 2. The expansion method of claim 1, further comprising thestep of adding anti-CD3 monoclonal antibody to the culture mediumwherein the concentration of anti-CD3 monoclonal antibody is at least1.0 ng/ml.
 3. The expansion method of claim 1, wherein said mammaliancell line comprises at least one cell type that is present at afrequency at least three times that found in human peripheral bloodmononuclear cells (human PBMCs).
 4. The expansion method of claim 1,wherein said T-cell-stimulatory component is an Fc-γ receptor.
 5. Theexpansion method of claim 1, wherein said T-cell-stimulatory componentis a cell adhesion-accessory molecule.
 6. The expansion method of claim1, wherein said T-cell-stimulatory component is a cytokine.
 7. Theexpansion method of claim 5, wherein said cell adhesion-accessorymolecule is selected from the group consisting of Class II MHC, Class IMHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, CD80, CD86,hyaluronate, and CD27 ligand.
 8. The expansion method of claim 1,wherein said T-cell-stimulatory component is a molecule that binds toCD21.
 9. The expansion method of claim 6, wherein said cytokine isselected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7,IL-12 and IL-15.
 10. The expansion method of claim 1, further comprisingthe step of adding a soluble T-cell-stimulatory factor to the culturemedium.
 11. The expansion method of claim 10, wherein said solubleT-cell-stimulatory factor is selected from the group consisting of acytokine, an antibody specific for a T cell surface component, and anantibody specific for a component capable of binding to a T cell surfacecomponent.
 12. The expansion method of claim 11, wherein said solubleT-cell-stimulatory factor is a cytokine selected from the groupconsisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.
 13. Theexpansion method of claim 11, wherein said soluble T-cell-stimulatoryfactor is an antibody specific for a T cell surface component, andwherein said T cell surface component is selected from the groupconsisting of CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44. 14.The expansion method of claim 11, wherein said solubleT-cell-stimulatory factor is an antibody specific for a componentcapable of binding to a T cell surface component, and wherein said Tcell surface component is selected from the group consisting of CD4,CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.
 15. The expansionmethod of claim 10, wherein said soluble T-cell-stimulatory factor is amolecule that binds to CD21.
 16. The expansion method of claim 15,wherein said molecule that binds to CD21 is an anti-CD21 antibody. 17.The expansion method of claim 1, further comprising the step of addingto the culture a multiplicity of peripheral blood mononuclear cells(PBMCs).
 18. The expansion method of claim 17, wherein the ratio ofPBMCs to initial T cells to be expanded is less than 40:1.
 19. Theexpansion method of claim 17, wherein the ratio of PBMCs to initial Tcells to be expanded is less than 10:1.
 20. The expansion method ofclaim 17, wherein the ratio of PBMCs to initial T cells to be expandedis less than 3:1.
 21. The expansion method of claim 1, furthercomprising the step of adding to the culture a multiplicity ofEBV-transformed lymphoblastoid cells (LCLs).
 22. The expansion method ofclaim 21, wherein the ratio of LCLs to initial T cells to be expanded isless than 10:1.
 23. The expansion method of claim 1, wherein the initialT lymphocyte population comprises at least one humanCD8+antigen-specific cytotoxic T lymphocyte (CTL).
 24. The expansionmethod of claim 1, wherein the initial T lymphocyte population comprisesat least one human CD4+antigen-specific helper T lymphocyte.
 25. Amethod for rapidly expanding an initial T lymphocyte population inculture medium in vitro, comprising the steps of: adding an initial Tlymphocyte population to a culture medium in vitro; adding to theculture medium a molecule that binds to CD21; and incubating theculture, thereby expanding the T lymphocyte population.
 26. The methodof claim 25, wherein said molecule that binds to CD21 is an anti-CD21antibody.